We report an outbreak of SARS-CoV-2 lineage alpha in gorillas and felid species in a zoo in Prague, Czech Republic. The course of illness and clinical signs are described, as are the results of characterization of these particular SARS-CoV-2 variants by next-generation sequencing and phylogenetic analysis. The putative transmission routes are also discussed.
- MeSH
- COVID-19 * MeSH
- Felidae * MeSH
- fylogeneze MeSH
- Hominidae * MeSH
- lidé MeSH
- SARS-CoV-2 genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Despite their widespread distribution, only a single genotype variant of clade 2.3.4.4b H5N1 influenza viruses has been found so far in Europe. Here, we report the detection of a new highly pathogenic avian influenza H5N1 genotype in geese and ducks from a backyard farm in the Czech Republic. Phylogenetic analysis revealed that the Czech H5N1 virus retained the A/Eurasian_Wigeon/Netherlands/1/2020-like backbone with an altered PB2 segment obtained from co-circulating low-pathogenic avian influenza viruses.
- MeSH
- fylogeneze MeSH
- genotyp MeSH
- husy virologie MeSH
- kachny virologie MeSH
- ptačí chřipka u ptáků * epidemiologie virologie MeSH
- virus chřipky A, podtyp H5N1 * genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Evropa MeSH
The mutual dependence of human and animal health is central to the One Health initiative as an integrated strategy for infectious disease control and management. A crucial element of the One Health includes preparation and response to influenza A virus (IAV) threats at the human-animal interface. The IAVs are characterized by extensive genetic variability, they circulate among different hosts and can establish host-specific lineages. The four main hosts are: avian, swine, human and equine, with occasional transmission to other mammalian species. The host diversity is mirrored in the range of the RT-qPCR assays for IAV detection. Different assays are recommended by the responsible health authorities for generic IAV detection in birds, swine or humans. In order to unify IAV monitoring in different hosts and apply the One Health approach, we developed a single RT-qPCR assay for universal detection of all IAVs of all subtypes, species origin and global distribution. The assay design was centred on a highly conserved region of the IAV matrix protein (MP)-segment identified by a comprehensive analysis of 99,353 sequences. The reaction parameters were effectively optimised with efficiency of 93-97% and LOD95% of approximately ten IAV templates per reaction. The assay showed high repeatability, reproducibility and robustness. The extensive in silico evaluation demonstrated high inclusivity, i.e. perfect sequence match in the primers and probe binding regions, established as 94.6% for swine, 98.2% for avian and 100% for human H3N2, pandemic H1N1, as well as other IAV strains, resulting in an overall predicted detection rate of 99% on the analysed dataset. The theoretical predictions were confirmed and extensively validated by collaboration between six veterinary or human diagnostic laboratories on a total of 1970 specimens, of which 1455 were clinical and included a diverse panel of IAV strains.
- MeSH
- chřipka lidská diagnóza virologie MeSH
- infekce viry z čeledi Orthomyxoviridae diagnóza virologie MeSH
- lidé MeSH
- nemoci prasat diagnóza virologie MeSH
- One Health MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- prasata MeSH
- ptačí chřipka u ptáků diagnóza virologie MeSH
- ptáci virologie MeSH
- reprodukovatelnost výsledků MeSH
- virus chřipky A, podtyp H1N1 genetika izolace a purifikace MeSH
- virus chřipky A, podtyp H3N2 genetika izolace a purifikace MeSH
- virus chřipky A genetika izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- MeSH
- COVID-19 MeSH
- SARS-CoV-2 * genetika izolace a purifikace MeSH
- sekvenování celého genomu metody statistika a číselné údaje MeSH
- Geografické názvy
- Česká republika MeSH
NRL pro chřipku a nechřipková respirační virová onemocnění, detekovala prvé 3 pozitivní případy v ČR 1. 3. 2020, Tyto 3 pozitivní případy byly odeslány ke konfirmaci do EU referenční laboratoře Charité v Berlíně, přesto ještě před oficiální konfirmací pozitivity, byla parciální sekvenací S genu potvrzena pozitivita přímo v la-boratoři. Shodou okolností právě u prvého záchytu jsme dokázali ve spolupráci s SVU Praha získat první českou celogenomovou sekvenci, která byla již 12. 3. 2020 přijata do databáze GISAID. Kmen se zařadil do skupiny „clade“ G, stejně jako další dva osekvenované materiály. Do této klády patří nejen většina italských záchytů viru, ale také většina záchytů z Evropy. Další 2 celogenomové sekvence byly získány v Charité a lze řící, že vyjma několika krátkých úseků, kde je čtení pro Oxford Nanopore technologii obtížné, jsou všechny 3 sekvence shodné, přestože se jedná o 3 nezávislé vstupy.Současně byla NRL úspěšná v izolaci 3 kmenů viru na VERO buňkách. V současnosti je v NRL prováděna především přímá laboratorní diagnostika nového pandemického viru SARS-CoV-2, doposud vyšetřila více než 4 000 pacientů.
The National Reference Laboratory for Influenza and Non-Influenza Viral Diseases (NRL) detected the first three COVID-19 cases in the Czech Republic on 1 March 2020. Their results were referred for confirmation to the EU reference laboratory Charité in Berlin. Before obtaining the official results, the NRL confirmed the COVID-19 positivity by partial S gene sequencing. Coincidentally, in collaboration with the State Veterinary Institute Prague, the first Czech whole genome sequence was obtained for the first virus detected and was submitted to the GISAID database on 12 March 2020. The strain was assigned to clade G, similarly to the other two strains sequenced. Clade G groups not only most Italian but also most European strains. The other two whole genome sequences were obtained from Charité. It can be stated that except for short nucleotide motifs, which are difficult to read by the Oxford Nanopore technology, all three sequences are identical although originating from three different inputs.The NRL also successfully isolated three strains of the virus on VERO cells. At present, the NRL performs mainly direct diagnosis of the new pandemic virus SARS-CoV-2. Over 4 000 patients have been tested to date by the NRL.
- MeSH
- Betacoronavirus * genetika izolace a purifikace MeSH
- koronavirové infekce diagnóza epidemiologie MeSH
- lidé MeSH
- sekvenování celého genomu * MeSH
- Check Tag
- lidé MeSH
- Geografické názvy
- Česká republika MeSH
The ongoing evolution of microbial pathogens represents a significant issue in diagnostic PCR/qPCR. Many assays are burdened with false negativity due to mispriming and/or probe-binding failures. Therefore, PCR/qPCR assays used in the laboratory should be periodically re-assessed in silico on public sequences to evaluate the ability to detect actually circulating strains and to infer potentially escaping variants. In the work presented we re-assessed a RT-qPCR assay for the universal detection of influenza A (IA) viruses currently recommended by the European Union Reference Laboratory for Avian Influenza. To this end, the primers and probe sequences were challenged against more than 99,000 M-segment sequences in five data pools. To streamline this process, we developed a simple algorithm called the SequenceTracer designed for alignment stratification, compression, and personal sequence subset selection and also demonstrated its utility. The re-assessment confirmed the high inclusivity of the assay for the detection of avian, swine and human pandemic H1N1 IA viruses. On the other hand, the analysis identified human H3N2 strains with a critical probe-interfering mutation circulating since 2010, albeit with a significantly fluctuating proportion. Minor variations located in the forward and reverse primers identified in the avian and swine data were also considered.
Ongoing evolution of viral pathogens is a significant issue in diagnostic virology employing TaqMan qPCR/RT-qPCR. Specific concerns are related to false negativity due to probe binding failure. One option for compensating for such deficiency is to integrate a second identically labelled probe in the assay. However, how this alteration influences the reaction parameters has not been comprehensively demonstrated. In the present study, we evaluate a TaqMan protocol using two identically labelled hydrolysis probes (simple, LNA (locked-nucleic-acid)) and MGB (minor-groove-binder) modified probes and combinations thereof in a single assay. Our results based on a synthetic amplicon suggest that the second probe does not compromise the TaqMan qPCR/RT-qPCR parameters, which repeatedly and reproducibly remained comparable to those of the corresponding single-probe assays, irrespective of the relative probe orientation, whether opposite or tandem, and probe modifications or combinations thereof. On the other hand, the second probe additively contributed to the overall fluorescence signal. The utility of the dual-probe approach was demonstrated on practical examples by using field specimens. We hope that the present study might serve as a theoretical basis for the development or improvement of TaqMan qPCR/RT-qPCR assays for the detection of highly variable nucleic acid templates.
- MeSH
- barvení a značení * MeSH
- DNA sondy metabolismus MeSH
- fluorescence MeSH
- hydrolýza MeSH
- kalibrace MeSH
- koně MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- nukleové kyseliny metabolismus MeSH
- psi MeSH
- reprodukovatelnost výsledků MeSH
- sekvence nukleotidů MeSH
- zvířata MeSH
- Check Tag
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Epidemická sezona 2014/2015 byla charakterizována dominancí subtypu H3N2. Cílem této studie je analýza genetické a antigenní heterogenity kmenů H3N2 detekovaných v České republice v období listopad 2014 až březen 2015 na úrovni molekuly hemaglutininu (HA) H3. Za tímto účelem byla provedena fylogenetická analýza reprezentativních sekvencí H3 HA a glykosylační status a klíčové antigenní mutace byly porovnány s vakcinálními kmeny pro roky 2014 a 2015 (A/Texas/50/2012 a A/Switzerland/9715293/2013) a následně vizualizovány v krystalografické struktuře proteinu H3. Molekulární analýza byla doplněna o výsledky hemaglutinačně inhibičního testu (HIT) s patnácti H3N2 kmeny s využitím antisér A/Texas/50/2012 a A/Switzerland/9715293/2013. Výsledky analýzy českých H3N2 kmenů cirkulujících v sezoně 2014/2015 slouží jako doplnění výsledů oficiálních institucí o data z konkrétní geografické oblasti.
The 2014/2015 influenza epidemic season was characterized by the predominance of the H3N2 subtype. The presented study investigated the genetic and antigenic heterogeneity of the H3N2 strains collected in the Czech Republic from November 2014 to March 2015. Phylogenetic analysis of the representative H3 hemagglutinin sequences was performed and the glycosylation status and crucial antigenic mutations were compared relative to the 2014 and 2015 vaccine strains (A/Texas/50/2012 and A/Switzerland/9715293/2013) and visualized in the H3 crystal structure. The molecular data were further supplemented by hemagglutination-inhibition test (HIT) results on fifteen H3N2 2014/2015 strains by using the A/Texas/50/2012 (H3N2) and A/Switzerland/9715293/13 (H3N2) antisera. Our data on the Czech H3N2 viruses from the 2014/2015 epidemic season could supplement the reports of official authorities with data from a particular geographical area.
- MeSH
- antigenní variace MeSH
- antigeny virové MeSH
- chřipka lidská virologie MeSH
- epidemie MeSH
- fylogeneze MeSH
- glykosylace MeSH
- hemaglutininové glykoproteiny viru chřipky * genetika MeSH
- lidé MeSH
- mutace MeSH
- sekvence aminokyselin MeSH
- sekvenční analýza proteinů MeSH
- testy inhibice hemaglutinace * MeSH
- vakcíny proti chřipce MeSH
- virus chřipky A, podtyp H3N2 * genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.
- MeSH
- DNA sondy chemie metabolismus MeSH
- fluoresceiny chemie MeSH
- fluorescenční barviva chemie MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- nukleové kyseliny metabolismus MeSH
- RNA virová metabolismus MeSH
- Taq-polymerasa metabolismus MeSH
- virus chřipky A genetika MeSH
- virus slintavky a kulhavky genetika MeSH
- změna skupenství MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The development of a diagnostic polymerase chain reaction (PCR) or quantitative PCR (qPCR) assay for universal detection of highly variable viral genomes is always a difficult task. The purpose of this chapter is to provide a guideline on how to align, process, and evaluate a huge set of homologous nucleotide sequences in order to reveal the evolutionarily most conserved positions suitable for universal qPCR primer and hybridization probe design. Attention is paid to the quantification and clear graphical visualization of the sequence variability at each position of the alignment. In addition, specific problems related to the processing of the extremely large sequence pool are highlighted. All of these steps are performed using an ordinary desktop computer without the need for extensive mathematical or computational skills.
