scholarly journals High-Throughput Fluorescent Assay for Inhibitor Screening of Proteases from RNA Viruses

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3792
Author(s):  
Bara Cihlova ◽  
Andrea Huskova ◽  
Jiri Böserle ◽  
Radim Nencka ◽  
Evzen Boura ◽  
...  
Keyword(s):  
Energy Transfer ◽  
High Throughput ◽  
Rna Viruses ◽  
Fluorescent Assay ◽  
Reaction Progress ◽  
Viral Proteases ◽  
Tick Borne Encephalitis ◽  
Spanish Flu ◽  
Tick Borne Encephalitis Virus ◽  
High Throughput Assay

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.

scholarly journals High-throughput fluorescent assay for inhibitor screening of proteases from RNA viruses

2020 ◽  
Author(s):  
Bara Cihlova ◽  
Andrea Huskova ◽  
Jiri Böserle ◽  
Radim Nencka ◽  
Evzen Boura ◽  
...  
Keyword(s):  
High Throughput ◽  
Rna Viruses ◽  
Fluorescent Proteins ◽  
Viral Disease ◽  
Fluorescent Assay ◽  
Viral Proteases ◽  
Tick Borne Encephalitis Virus ◽  
New Treatments ◽  
Influenza Outbreaks

AbstractSpanish flu and other influenza outbreaks, the recent Zika epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases that are caused by RNA viruses. Perhaps less well known yet dangerous RNA viruses cause deadly diseases such as polio, Ebola, measles, rubella, yellow fever, dengue fever and many others. To combat a particular viral disease by diminishing its spread and number of fatal cases, effective vaccines and antivirals are indispensable. Therefore, quick access to the means of discovery of new treatments for any epidemic outbreak is of great interest and in vitro biochemical assays are the basis of drug discovery. The recent outbreak of the coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands an affordable and reliable assay for testing antivirals. Here, we developed a quick and inexpensive high-throughput fluorescent assay to test inhibitors of viral proteases. Accordingly, we employed this assay to sample inhibitors for papain-like protease from SARS-CoV-2. In addition, we validated this assay for screening inhibitors of flaviviral protease from the tick-borne encephalitis virus to emphasize a broad range of applications of our approach. This fluorescent high-throughput assay is based on fluorescent energy transfer (FRET) between two distinct fluorescent proteins (eGFP and mCherry) connected via a substrate polypeptide. When the substrate is cleaved, FRET is abolished and the change in fluorescence corresponds to reaction progress. Our data show that this assay can be used for testing the inhibitors in the 96 or 384 well plates format with robust and reproducible outcomes.

scholarly journals High-throughput sequencing of two European strains of tick-borne encephalitis virus (TBEV), Hochosterwitz and 1993/783

2021 ◽  
Vol 12 (1) ◽  
pp. 101557
Author(s):  
Katrine M. Paulsen ◽  
Alaka Lamsal ◽  
Srijana Bastakoti ◽  
John H.-O. Pettersson ◽  
Benedikte N. Pedersen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Encephalitis Virus ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus

TBE in South Korea

2019 ◽  
Author(s):  
Joon Young Song
Keyword(s):  
South Korea ◽  
Human Case ◽  
Encephalitis Virus ◽  
Surveillance Studies ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus

Although no human case of tick-borne encephalitis (TBE) has been documented in South Korea to date, surveillance studies have been conducted to evaluate the prevalence of tick-borne encephalitis virus (TBEV) in wild ticks.

TBE in Slovakia

2019 ◽  
Author(s):  
Jana Kerlik
Keyword(s):  
Encephalitis Virus ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus

The former Czechoslovak Republic was one of the first countries in Europe where the tick-borne encephalitis virus (TBEV) was identified.

TBE in Sweden

2020 ◽  
Keyword(s):  
Genetic Characterization ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Second Phase ◽  
Serum Samples ◽  
Tick Borne Encephalitis ◽  
Specific Immunoglobulin ◽  
Tick Borne Encephalitis Virus ◽  
First Time

Tick-borne encephalitis virus (TBEV) was isolated for the first time in Sweden in 1958 (from ticks and from 1 tick-borne encephalitis [TBE] patient).1 In 2003, Haglund and colleagues reported the isolation and antigenic and genetic characterization of 14 TBEV strains from Swedish patients (samples collected 1991–1994).2 The first serum sample, from which TBEV was isolated, was obtained 2–10 days after onset of disease and found to be negative for anti-TBEV immunoglobulin M (IgM) by enzyme-linked immunosorbent assay (ELISA), whereas TBEV-specific IgM (and TBEV-specific immunoglobulin G/cerebrospinal fluid [IgG/CSF] activity) was demonstrated in later serum samples taken during the second phase of the disease.

Chapter 11: General epidemiology of TBE

2020 ◽  
Keyword(s):  
Eastern Siberia ◽  
Genetic Lineages ◽  
Phylogenetic Studies ◽  
Tick Borne Encephalitis ◽  
Natural Foci ◽  
Genetic Sequences ◽  
Tick Borne Encephalitis Virus ◽  
Almost All ◽  
Far Eastern ◽  
Reservoir Hosts

Tick-borne encephalitis virus (TBEV) exists in natural foci, which are areas where TBEV is circulating among its vectors (ticks of different species and genera) and reservoir hosts (usually rodents and small mammals). Based on phylogenetic studies, four TBEV subtypes (Far-Eastern, Siberian, European, Baikalian) and two putative subtypes (Himalayan and “178-79” group) are known. Within each subtype, some genetic lineages are described. The European subtype (TBEV-EU) (formerly known also as the “Western subtype”) of TBEV is prevalent in Europe, but it was also isolated in Western and Eastern Siberia in Russia and South Korea. The Far-Eastern subtype (TBEV-FE) was preferably found in the territory of the far-eastern part of Eurasia, but some strains were isolated in other regions of Eurasia. The Siberian (TBEV-SIB) subtype is the most common and has been found in almost all TBEV habitat areas. The Baikalian subtype is prevalent around Lake Baikal and was isolated several times from ticks and rodents. In addition to the four TBEV subtypes, one single isolate of TBEV (178-79) and two genetic sequences (Himalayan) supposed to be new TBEV subtypes were described in Eastern Siberia and China. The data on TBEV seroprevalence in humans and animals can serve as an indication for the presence or absence of TBEV in studied area.

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