Here, we present a comprehensive analysis of the H5N8/H5N5 highly pathogenic avian influenza (HPAI) virus strains detected in the Czech Republic during an outbreak in 2017. Network analysis of the H5 Hemagglutinin (HA) from 99% of the outbreak localities suggested that the diversity of the Czech H5N8/H5N5 viruses was influenced by two basic forces: local microevolution and independent incursions. The geographical occurrence of the central node H5 HA sequences revealed three eco-regions, which apparently played an important role in the origin and further spread of the local H5N8/HPAI variants across the country. A plausible explanation for the observed pattern of diversity is also provided.
- MeSH
- epidemický výskyt choroby MeSH
- fylogeneze MeSH
- genetická variace MeSH
- hemaglutininové glykoproteiny viru chřipky genetika MeSH
- molekulární evoluce * MeSH
- ptačí chřipka u ptáků epidemiologie virologie MeSH
- ptáci klasifikace virologie MeSH
- virulence MeSH
- virus chřipky A, podtyp H5N8 klasifikace genetika izolace a purifikace patogenita MeSH
- virus chřipky A klasifikace genetika izolace a purifikace patogenita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika epidemiologie MeSH
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
Influenza A virus (IAV) in wild bird reservoir hosts is characterized by the perpetuation in a plethora of subtype and genotype constellations. Multiyear monitoring studies carried out during the last two decades worldwide have provided a large body of knowledge regarding the ecology of IAV in wild birds. Nevertheless, other issues of avian IAV evolution have not been fully elucidated, such as the complexity and dynamics of genetic interactions between the co-circulating IAV genomes taking place at a local-scale level or the phenomenon of frozen evolution. We investigated the IAV diversity in a mallard population residing in a single pond in the Czech Republic. Despite the relative small number of samples collected, remarkable heterogeneity was revealed with four different IAV subtype combinations, H6N2, H6N9, H11N2, and H11N9, and six genomic constellations in co-circulation. Moreover, the H6, H11, and N2 segments belonged to two distinguishable sub-lineages. A reconstruction of the pattern of genetic reassortment revealed direct parent-progeny relationships between the H6N2, H11N9 and H6N9 viruses. Interestingly the IAV, with the H6N9 subtype, was re-detected a year later in a genetically unchanged form in the close proximity of the original sampling locality. The almost absolute nucleotide sequence identity of all the respective genomic segments between the two H6N9 viruses indicates frozen evolution as a result of prolonged conservation in the environment. The persistence of the H6N9 IAV in various abiotic and biotic environmental components was also discussed.
The objective of our study was to provide a genotype analysis of H7N7 and H7N9 influenza A viruses (IAV) and infer their relationships to co-circulating non-H7 IAV genomes. The H7N7 strains were collected in central Europe (Hungary-1, Czech Republic-1, Slovenia-1 and Poland-4) and the H7N9 in the Czech Republic and Spain between 2007 and 2011. Hand in hand with this effort, a novel IAV genotype visualization approach called digital genotyping was developed. This approach relies on phylogenetic data summarization and transformation into a pixel array called a segment identity matrix. The digital genotyping revealed a complicated genetic interplay between the H7 and co-circulating non-H7 IAV genotypes. At the H7 IAV level the most obvious relationships were observed between one Polish H7N7/446/09 and Czech H7N7/11 viruses which, despite the special and temporal distance of 800 km and 15 months, retained at least 6/8 genome segments. Close relationships were also observed between the Czech H7N9, Polish and Slovenian H7N7 on one hand and Hungarian and Slovenian H7N7 isolates on the other. In addition the former genomes exhibited close interplays with the Czech H6N2/09 and H11N9/10-like viruses. The Czech and Spanish H7N9 genomes were completely different and 6/8 of the Czech H7N9-like segments were traced to either the Czech H3N8/07, H11N9/09 and Polish H7N7/09-like viruses. The results of digital genotyping correlated with the previous observations obtained on the Polish H7N7 isolates. As was demonstrated, the digital genotyping provides a well-arranged and easily interpretable output and may serve as an alternative genotyping tool useful for handling and analysing even a large panel of IAV genomes.
- MeSH
- fylogeneze MeSH
- genotyp MeSH
- molekulární epidemiologie metody MeSH
- molekulární sekvence - údaje MeSH
- molekulární typizace metody MeSH
- ptačí chřipka u ptáků epidemiologie virologie MeSH
- ptáci MeSH
- sekvenční analýza DNA MeSH
- shluková analýza MeSH
- virologie metody MeSH
- virus chřipky A klasifikace genetika izolace a purifikace MeSH
- výpočetní biologie metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Evropa MeSH
The objective of our study was to develop and evaluate a TaqMan real-time RT-PCR (RRT-PCR) assay for universal detection of influenza A (IA) viruses. The primers and LNA-modified octanucleotide probe were selected to correspond to extremely conserved regions of the membrane protein (MP) segment identified by a comprehensive bioinformatics analysis including 10,405 IA viruses MP sequences, i.e., all of the sequences of the Influenza Virus Sequence database collected as of August 20, 2009. The RRT-PCR has a detection limit of approximately five copies of target RNA/reaction and excellent reaction parameters tested in four IA viruses reference laboratories. The inclusivity of the assay was estimated at both the bioinformatic and the experimental level. Our results predicted that this RRT-PCR assay was able to detect 99.5% of known human IA virus strains, 99.84% of pandemic influenza A (H1N1) strains, 99.75% of avian strains, 98.89% of swine strains, 98.15% of equine strains, and 100% of influenza A viruses of other origin.
In order to determine the actual prevalence of avian influenza viruses (AIV) in wild birds in the Czech Republic extensive surveillance was carried out between January and April 2006. A total of 2101 samples representing 61 bird species were examined for the presence of influenza A by using PCR, sequencing and cultivation on chicken embryos. AIV subtype H5N1 was detected in 12 Mute swans (Cygnus olor). The viruses were determined as HPAI (highly pathogenic avian influenza) and the hemagglutinin sequence was closely similar to A/mallard/Italy/835/06 and A/turkey/Turkey/1194/05. Following the first H5N1 case, about 300 wild birds representing 33 species were collected from the outbreak region and tested for the presence of AIV without any positive result. This is the first report of highly pathogenic avian influenza subtype H5N1 in the Czech Republic. The potential role of swan as an effective vector of avian influenza virus is also discussed.
- MeSH
- Anseriformes virologie MeSH
- divoká zvířata MeSH
- fylogeneze MeSH
- kuřecí embryo MeSH
- molekulární sekvence - údaje MeSH
- prevalence MeSH
- ptačí chřipka u ptáků epidemiologie virologie MeSH
- surveillance populace MeSH
- virus chřipky A, podtyp H5N1 izolace a purifikace klasifikace patogenita MeSH
- zvířata MeSH
- Check Tag
- kuřecí embryo MeSH
- zvířata MeSH
- Geografické názvy
- Česká republika MeSH
- MeSH
- Anseriformes * MeSH
- ptačí chřipka u ptáků epidemiologie virologie MeSH
- virus chřipky A, podtyp H5N1 patogenita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- dopisy MeSH
- Geografické názvy
- Česká republika MeSH
