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2.
Artigo em Inglês | MEDLINE | ID: mdl-34205104

RESUMO

Acute febrile illnesses occur frequently in Guinea. Acute fever itself is not a unique, hallmark indication (pathognomonic sign) of any one illness or disease. In the infectious disease context, fever's underlying cause can be a wide range of viral or bacterial pathogens, including the Ebola virus. In this study, molecular and serological methods were used to analyze samples from patients hospitalized with acute febrile illness in various regions of Guinea. This analysis was undertaken with the goal of accomplishing differential diagnosis (determination of causative pathogen) in such cases. As a result, a number of pathogens, both viral and bacterial, were identified in Guinea as causative agents behind acute febrile illness. In approximately 60% of the studied samples, however, a definitive determination could not be made.


Assuntos
Técnicas de Laboratório Clínico , Febre , Diagnóstico Diferencial , Febre/diagnóstico , Febre/etiologia , Guiné/epidemiologia , Humanos
3.
J Med Virol ; 93(3): 1694-1701, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32966645

RESUMO

Coronavirus disease 2019 (COVID-19) has become pandemic since March 11, 2020. Thus, development and integration in clinics of fast and sensitive diagnostic tools are essential. The aim of the study is a development and evaluation of a one-step quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay (COVID-19 Amp) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection with an armored positive control and internal controls constructed from synthetic MS2-phage-based RNA particles. The COVID-19 Amp assay limit of detection was 103 copies/ml, the analytical specificity was 100%. A total of 109 biological samples were examined using COVID-19 Amp and World Health Organization (WHO)-based assay. Discordance in nine samples was observed (negative by the WHO-based assay) and discordant samples were retested as positive according to the results obtained from the Vector-PCRrv-2019-nCoV-RG assay. The developed COVID-19 Amp assay has high sensitivity and specificity, includes virus particles-based controls, provides the direct definition of the SARS-CoV-2 RdRp gene partial sequence, and is suitable for any hospital and laboratory equipped for RT-qPCR.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Testes Diagnósticos de Rotina , Feminino , Genoma Viral/genética , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Adulto Jovem
4.
Ticks Tick Borne Dis ; 12(2): 101612, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33291056

RESUMO

Wad Medani virus (WMV) belongs to the genus Orbivirus and is a poorly studied arbovirus with unclear medical significance. Presently, a limited number of WMV strains are characterized and available in NCBI GenBank, some isolated many years ago. A new WMV strain was isolated in 2012 from Dermacentor nuttalli ticks collected from sheep in the Tuva Republic, Russia, and sequenced using high-throughput methods. Complete coding sequences were obtained revealing signs of multiple intersegment reassortments. These point to a high variability potential in WMV that may lead to the formation of strains with novel properties. These new data on WMV can promote better understanding of: ecological features of its circulation; relationships within the genus Orbivirus; and the medical significance of the virus.

5.
Artigo em Inglês | MEDLINE | ID: mdl-32545855

RESUMO

This article describes a lethal case of leptospirosis that occurred in Southern Russia. The Leptospira strain was isolated and characterized using a microscopic agglutination test, MALDI-TOF mass spectrometry, targeted PCR, and high-throughput sequencing. We show that molecular and mass-spectrometry methods can be an alternative to conventional methods of leptospirosis diagnostics and Leptospira study, which require highly qualified staff and can be performed only at specialized laboratories. We also report the first whole genome of L. interrogans isolated in Russia.


Assuntos
Leptospira interrogans , Leptospira , Leptospirose , Adolescente , Testes de Aglutinação , Humanos , Federação Russa
6.
Ticks Tick Borne Dis ; 11(2): 101333, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31787560

RESUMO

Kemerovo virus (KEMV) is a member of the Great Island virus genetic group, belonging to the tick-borne arboviruses of the genus Orbivirus within the family Reoviridae. Nine strains of KEMV, which were isolated from various locations in Russia, were sequenced by high-throughput sequencing to study their intraspecific diversity and the interspecific relationships of viruses within the Great Island genetic group. For the first time, multiple reassortment within KEMV was reliably demonstrated. Different types of independently emerged alternative reading frames in segment 9 and heterogeneity of the viral population in one of the KEMV strains were found. The hypothesis of the role of an alternative open reading frame (ORF) in segment 9 in KEMV cellular tropism was not confirmed in this study.


Assuntos
Variação Genética , Genoma Viral , Orbivirus/genética , Filogenia , Federação Russa , Análise de Sequência de DNA
7.
Methods Mol Biol ; 2063: 181-188, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31667771

RESUMO

This chapter reports a library preparation protocol for efficient high-throughput sequencing of double-stranded RNA viruses. The protocol consists of four main steps, viz., enzyme treatment, precipitation using lithium chloride, full-length amplification of cDNAs, and tailing adapters for high-throughput sequencing. This protocol will be useful for all double-stranded RNA viruses and for all of the high-throughput sequencing platforms.


Assuntos
Genoma Viral/genética , Biblioteca Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Vírus de RNA/genética , RNA de Cadeia Dupla/genética , Primers do DNA/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Orbivirus/genética , Reação em Cadeia da Polimerase , RNA Viral/genética
8.
J Virol Methods ; 271: 113674, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31170468

RESUMO

Lassa fever is a severe viral hemorrhagic illness caused by Lassa virus. Based on estimates, the number of LASV infections ranges from 300,000 to 500,000 cases in endemic areas with a fatality rate of 1%. Development of fast and sensitive tools for the control and prevention of Lassa virus infection as well as for clinical diagnostics of Lassa fever are crucial. Here we reported development and evaluation of a one-step quantitative RT-qPCR assay for the Lassa virus detection - LASV-Fl. This assay is suitable for the detection of lineages I-IV of Lassa virus. The limit of detection of the assay ranged from 103 copies/ml to 105 copies/ml and has 96.4% diagnostic sensitivity, whereas analytical and diagnostic specificities both were 100%. Serum, whole blood and tissue are suitable for use with the assay. The assay contains all the necessary components to perform the analysis, including an armored positive control (ARC+) and an armored internal control (IC). The study was done during the mission of specialized anti-epidemic team of the Russian Federation (SAET) in the Republic of Guinea in 2015-2018. Based on sequencing data, LASV-specific assay was developed using synthetic MS2-phage-based armored RNA particles, RNA from Lassa virus strain Josiah, and further, evaluated in field conditions using samples from patients and Mastomys natalensis rodents.


Assuntos
Febre Lassa/diagnóstico , Vírus Lassa/isolamento & purificação , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adolescente , Adulto , Animais , Criança , Primers do DNA/genética , Sondas de DNA/genética , Feminino , Guiné , Humanos , Febre Lassa/sangue , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Murinae/virologia , Sensibilidade e Especificidade , Adulto Jovem
9.
Methods Mol Biol ; 1973: 281-297, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016709

RESUMO

We developed a new technique suitable for improved detection of low-copy dsRNA using modified oligonucleotides as primers in RT-qPCR. Insertion of G8AE-clamp residues into primers significantly improves thermal stability of duplexes with RNA without decrease of hybridization selectivity. The applicability of modified primers is demonstrated for detection of low-copy Kemerovo virus dsRNA.


Assuntos
Primers do DNA/química , Oligonucleotídeos/química , RNA de Cadeia Dupla/análise , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA Viral/química , RNA Viral/genética
10.
Ticks Tick Borne Dis ; 10(2): 269-279, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30448254

RESUMO

Paramushir virus belongs to Sakhalin virus genogroup within Orthonairovirus genus and is one of the poorly studied viruses with unknown pathogenicity. At the moment, only one nearly complete sequence of Paramushir virus genome, isolated in 1972, is available. Two new strains of PARV were isolated in 2015 from a sample collected at the Tyuleniy Island in the Okhotsk Sea and sequenced using a combination of high throughput sequencing and specific multiplex PCR. Both strains are closely related to the early sequenced PARV strain LEIV-1149 K. The signs of intersegment reassortment and probable recombination were revealed, which point to a high variability potential of Paramushir virus and may lead to the formation of strains with novel properties, different from those of the predecessors. The new data regarding Paramushir virus can promote a better understanding of the diversity and relations within Orthonairovirus genus and help define intragenic demarcation criteria, which have not yet been established.


Assuntos
Nairovirus/genética , Filogenia , Carrapatos/virologia , Animais , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Ilhas , Reação em Cadeia da Polimerase Multiplex , Nairovirus/isolamento & purificação , RNA Viral/isolamento & purificação , Vírus Reordenados/genética , Vírus Reordenados/isolamento & purificação , Recombinação Genética , Federação Russa
11.
Adv Virol ; 2018: 3248285, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30158979

RESUMO

Advances in the next generation sequencing (NGS) technologies have significantly increased our ability to detect new viral pathogens and systematically determine the spectrum of viruses prevalent in various biological samples. In addition, this approach has also helped in establishing the associations of viromes with many diseases. However, unlike the metagenomic studies using 16S rRNA for the detection of bacteria, it is impossible to create universal oligonucleotides to target all known and novel viruses, owing to their genomic diversity and variability. On the other hand, sequencing the entire genome is still expensive and has relatively low sensitivity for such applications. The existing approaches for the design of oligonucleotides for targeted enrichment are usually involved in the development of primers for the PCR-based detection of particular viral species or genera, but not for families or higher taxonomic orders. In this study, we have developed a computational pipeline for designing the oligonucleotides capable of covering a significant number of known viruses within various taxonomic orders, as well as their novel variants. We have subsequently designed a genus-specific oligonucleotide panel for targeted enrichment of viral nucleic acids in biological material and demonstrated the possibility of its application for virus detection in bird samples. We have tested our panel using a number of collected samples and have observed superior efficiency in the detection and identification of viral pathogens. Since a reliable, bioinformatics-based analytical method for the rapid identification of the sequences was crucial, an NGS-based data analysis module was developed in this study, and its functionality in the detection of novel viruses and analysis of virome diversity was demonstrated.

12.
J Gen Virol ; 99(2): 240-245, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29393021

RESUMO

We studied minor variants within two tick-borne encephalitis virus (TBEV) populations with a common ancestor: the mouse brain-adapted variant EK-328c and the tick-adapted variant M. High-throughput sequencing with custom amplicons from RT-PCR viral RNA was performed on Illumina MiSeq 2*250 paired-end v2 chemistry. Using the LowFreq program (default settings) and Sanger-sequenced consensus as a reference, variants with an abundance of 1 % and above within the studied populations were identified. Using the obtained data in the context of our previous studies, we concluded that TBEV variants, which are different from the major population phenotype and can become a major part of the viral population under favourable environmental conditions, can exist at abundances of less than 1 % in the long-term. The comparison of our data with the literature allowed us to conclude that the laboratory variant EK-328c and variant M have similar SNV counts to TBEV variants from natural populations and some fast-evolving RNA viruses.


Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/genética , Encefalite Transmitida por Carrapatos/virologia , Animais , Heterogeneidade Genética , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , RNA Viral/genética , Análise de Sequência de RNA
13.
Health Secur ; 16(1): 14-21, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29350545

RESUMO

Filoviruses are important etiological agents of emergent diseases with high mortality rates. Traditionally, filovirus fever diseases have primarily been a burden of African countries; however, global interconnectedness has increased the probability of the worldwide spread of filoviruses. Therefore, national healthcare organizations need tools for managing filovirus risk, including diagnostic kits based on real-time reverse transcription PCR (RT-qPCR), as this is the most suitable method for diagnosing filovirus fever diseases. Here we describe a real-time RT-qPCR assay for filovirus detection. This assay is a further development of our previously reported EBOV (Zaire)-Fl kit. Two sets (FiloA-Fl and FiloB-Fl) of real-time RT-qPCR assays for the detection of filoviruses were developed and evaluated using armored RNA phage particles (ARs) as positive controls. The limit of detection of the assay was 5x102 copies/ml of the AR-positive control for the FiloA-Fl set and 5x103 copies/ml of the AR-positive control for the FiloB-Fl set. Our assay provides a rapid and sensitive tool for detecting filoviruses. The high specificity and sensitivity of the assay make it useful for clinical and epidemiologic investigations in the field of filovirus fever diseases and their etiological agents.


Assuntos
Ebolavirus/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Ebolavirus/genética , Doença pelo Vírus Ebola/diagnóstico , Doença pelo Vírus Ebola/virologia , Humanos , Testes Imediatos , Sensibilidade e Especificidade
15.
Bioorg Med Chem ; 25(14): 3597-3605, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28396019

RESUMO

Nowadays modified oligonucleotides are widely used in diagnostics and as novel therapeutics. Introduction of modified or unnatural residues into oligonucleotides allows fine tuning of their binding properties to complementary nucleic acids and leads to improved stability both in vitro and in vivo. Previously it was demonstrated that insertion of phenoxazine nucleotides with various groups in C9-position into oligonucleotides leads to a significant increase of duplex stability with complementary DNA and RNA. Here the synthesis of a novel G-clamp nucleoside analogue (G8AE-clamp) bearing 2-aminoethyl tether at C8-atom is presented. Introduction of such modified residues into oligonucleotides lead to enhanced specificity of duplex formation towards complementary DNA and RNA targets with increased thermal and 3'-exonuclease stability. According to CD-spectroscopy studies G8AE-clamp does not substantially disrupt helix geometry. Primers containing G8AE-clamp demonstrated superior sensitivity in qPCR detection of dsRNA of Kemerovo virus in comparison to native oligonucleotides.


Assuntos
Guanosina/análogos & derivados , Oligonucleotídeos/síntese química , Orbivirus/genética , Oxazinas/química , RNA Viral/metabolismo , Dicroísmo Circular , Exonucleases/metabolismo , Guanosina/metabolismo , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Oligonucleotídeos/química , RNA de Cadeia Dupla/análise , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real
16.
PLoS One ; 12(2): e0171855, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28225771

RESUMO

Near complete rabies virus N gene sequences (1,110 nt) were determined for 82 isolates obtained from different regions of Russia between 2008 and 2016. These sequences were analyzed together with 108 representative GenBank sequences from 1977-2016 using the Bayesian coalescent approach. The timing of the major evolutionary events was estimated. Most of the isolates represented the steppe rabies virus group C, which was found over a vast geographic region from Central Russia to Mongolia and split into three groups (C0-C2) with discrete geographic prevalence. A single strain of the steppe rabies virus lineage was isolated in the far eastern part of Russia (Primorsky Krai), likely as a result of a recent anthropogenic introduction. For the first time the polar rabies virus group A2, previously reported in Alaska, was described in the northern part of European Russia and at the Franz Josef Land. Phylogenetic analysis suggested that all currently circulating rabies virus groups in the Russian Federation were introduced within the few last centuries, with most of the groups spreading in the 20th century. The dating of evolutionary events was highly concordant with the historical epidemiological data.


Assuntos
Genoma Viral , Vírus da Raiva/genética , Filogenia , Raiva/virologia , Vírus da Raiva/isolamento & purificação , Federação Russa
17.
Genome Announc ; 4(5)2016 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-27789645

RESUMO

Human adenovirus 7 (hAdv7) 19BOVLB/Volgograd/Rus/2014 was isolated from the autopsy material from an adult with fatal pneumonia in Volgograd, Russia, in March 2014. Whole-genome sequencing of the virus isolate was performed.

19.
Genome Announc ; 4(4)2016 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-27389270

RESUMO

We report here the complete genome sequence (GenBank KP997032) of rabies virus strain RABV/Ursus arctos/Russia/Primorye/PO-01/2014, isolated in November 2014 from a brown bear (Ursus arctos) that attacked a person in Primorsky Krai (Russian Federation). This strain was clustered into the Eurasian genetic subgroup of genotype 1 (street rage).

20.
Vopr Virusol ; 61(5): 235-40, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-29323857

RESUMO

To improve the diagnosis, surveillance, and control for the rabies virus, a kit for hybridization-triggered fluorescence detection of rabies virus DNA by the RT-PCR technique was developed and evaluated. The analytical sensitivity of the kit was 4*10 GE per ml. High specificity of the kit was shown using representative sampling of viral, bacterial, and human nucleic acids.


Assuntos
RNA Viral/genética , Vírus da Raiva/genética , Raiva/epidemiologia , Raiva/veterinária , Reação em Cadeia da Polimerase em Tempo Real/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Animais , Gatos , Primers do DNA/síntese química , Primers do DNA/genética , Cervos/virologia , Cães , Raposas/virologia , Humanos , Raiva/diagnóstico , Raiva/transmissão , Vírus da Raiva/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Federação Russa/epidemiologia , Sensibilidade e Especificidade
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