High pathogenic avian influenza A(H5) viruses of clade 2.3.4.4b in Europe-Why trends of virus evolution are more difficult to predict.

Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario.

Enzootic Circulation, Massive Gull Mortality and Poultry Outbreaks during the 2022/2023 High-Pathogenicity Avian Influenza H5N1 Season in the Czech Republic.

In 2022/2023, Europe experienced its third consecutive season of high-pathogenicity avian influenza. During this period, the Czech Republic was again severely affected. For the first time, the number of culled birds approached one million, which was three times higher than in previous seasons. In parallel to the outbreaks in poultry, mass die-offs of gulls were also observed. In the present study, we performed whole-genome sequencing and phylogenetic analysis of 137 H5N1 strains collected in the Czech Republic in 2022/2023 (94.6% of all outbreaks or locations). The analysis revealed four distinct genotypes: AB, CH, BB and AF. Phylogenetic analysis suggested that the AF genotype persisted from the previous H5N1 season without reassortment. In addition, the genotype BB, which was detected mainly in gulls, showed a noticeable strain diversity at the local level. This virus was also responsible for a single outbreak in commercially bred turkeys. Finally, an interesting spatio-temporal cluster with three co-circulating H5N1 genotypes, AB, CH and AF, was identified with no evidence of intrasubtype reassortment. Highly sensitive molecular surveillance and the timely sharing of genomic sequences and associated metadata could greatly assist in tracking the spread and detecting molecular changes associated with the increased virulence of this potentially zoonotic pathogen.

Genotype Diversity, Wild Bird-to-Poultry Transmissions, and Farm-to-Farm Carryover during the Spread of the Highly Pathogenic Avian Influenza H5N1 in the Czech Republic in 2021/2022.

In 2021/2022, the re-emergence of highly pathogenic avian influenza (HPAI) occurred in Europe. The outbreak was seeded from two sources: resident and reintroduced viruses, which is unprecedented in the recorded history of avian influenza. The dominant subtype was H5N1, which replaced the H5N8 subtype that had predominated in previous seasons. In this study, we present a whole genome sequence and a phylogenetic analysis of 57 H5N1 HPAI and two low pathogenic avian influenza (LPAI) H5N1 strains collected in the Czech Republic during 2021/2022. Phylogenetic analysis revealed close relationships between H5N1 genomes from poultry and wild birds and secondary transmission in commercial geese. The genotyping showed considerable genetic heterogeneity among Czech H5N1 viruses, with six different HPAI genotypes, three of which were apparently unique. In addition, second-order reassortment relationships were observed with the direct involvement of co-circulating H5N1 LPAI strains. The genetic distance between Czech H5N1 HPAI and the closest LPAI segments available in the database illustrates the profound gaps in our knowledge of circulating LPAI strains. The changing dynamics of HPAI in the wild may increase the likelihood of future HPAI outbreaks and present new challenges in poultry management, biosecurity, and surveillance.

Retrospective Analysis Revealed an April Occurrence of Monkeypox in the Czech Republic.

Herein, we present our findings of an early appearance of the Monkeypox virus in Prague, Czech Republic. A retrospective analysis of biological samples, carried out on the 28th of April, revealed a previously unrecognized case of Monkeypox virus (MPxV) infection. Subsequent data analysis confirmed that the virus strain belongs to the ongoing outbreak. Combined with clinical and epidemiological investigations, we extended the roots of the current outbreak at least back to 16th of April, 2022.

Genotype Uniformity, Wild Bird-to-Poultry Transmissions, and Farm-to-Farm Carryover during the Spread of the Highly Pathogenic Avian Influenza H5N8 in the Czech Republic in 2021.

In 2020-2021, the second massive dissemination of a highly pathogenic avian influenza of the H5Nx subtype occurred in Europe. During this period, the virus caused numerous outbreaks in poultry, including in the Czech Republic. In the present study, we provide an insight into the genetic variability of the Czech/2021 (CZE/2021) H5N8 viruses to determine the relationships between strains from wild and domestic poultry and to infer transmission routes between the affected flocks of commercial poultry. For this purpose, whole genome sequencing and phylogenetic analysis of 70 H5N8 genomes representing 79.7% of the cases were performed. All CZE/2021 H5N8 viruses belonged to the 2.3.4.4b H5 lineage and circulated without reassortment, retaining the A/chicken/Iraq/1/2020 H5N8-like genotype constellation. Phylogenetic analysis suggested the frequent local transmission of H5N8 from wild birds to backyard poultry and extensive spread among commercial poultry farms. In addition, the analysis suggested one cross-border transmission event. Indirect transmission via contaminated materials was considered the most likely source of infection. Improved biosecurity and increased collaboration between field veterinarians and the laboratory are essential to limit the local spread of the virus and to reveal and interrupt critical routes of infection.

Has Epizootic Become Enzootic? Evidence for a Fundamental Change in the Infection Dynamics of Highly Pathogenic Avian Influenza in Europe, 2021.

Phylogenetic evidence from the recent resurgence of high-pathogenicity avian influenza (HPAI) virus subtype H5N1, clade 2.3.4.4b, observed in European wild birds and poultry since October 2021, suggests at least two different and distinct reservoirs. We propose contrasting hypotheses for this emergence: (i) resident viruses have been maintained, presumably in wild birds, in northern Europe throughout the summer of 2021 to cause some of the outbreaks that are part of the most recent autumn/winter 2021 epizootic, or (ii) further virus variants were reintroduced by migratory birds, and these two sources of reintroduction have driven the HPAI resurgence. Viruses from these two principal sources can be distinguished by their hemagglutinin genes, which segregate into two distinct sublineages (termed B1 and B2) within clade 2.3.4.4b, as well as their different internal gene compositions. The evidence of enzootic HPAI virus circulation during the summer of 2021 indicates a possible paradigm shift in the epidemiology of HPAI in Europe.

Reverse-zoonotic transmission of SARS-CoV-2 lineage alpha (B.1.1.7) to great apes and exotic felids in a zoo in the Czech Republic.

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.

A new clade 2.3.4.4b H5N1 highly pathogenic avian influenza genotype detected in Europe in 2021.

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.

A universal RT-qPCR assay for "One Health" detection of influenza A viruses.

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.

In silico re-assessment of a diagnostic RT-qPCR assay for universal detection of Influenza A viruses.

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.

Circulation of influenza A and B in the Czech Republic from 2000-2001 to 2015-2016.

BACKGROUND: To improve national influenza vaccination recommendations, additional data on influenza A and B virus circulation are needed. Here, we describe the circulation of influenza A and B in the Czech Republic during 16 seasons. METHODS: This was a retrospective analysis of data collected from the 2000-2001 to 2015-2016 influenza seasons by the Czech Republic national influenza surveillance network. Influenza was confirmed and viral isolates subtyped by virological assays followed by antigen detection or by reverse transcriptase-polymerase chain reaction. RESULTS: Of 16,940 samples collected, 5144 (30.4%) were influenza-positive. Influenza A represented 78.6% of positive cases overall and accounted for more than 55.0% of all influenza cases in every season, except for 2005-2006 (6.0%). Both A/H1N1 and A/H3N2 were detected in most seasons, except for 2001-2002 and 2003-2004 (only A/H3N2), and 2007-2008 and 2009-2010 (only A/H1N1). Influenza B represented 21.4% of positive cases overall (range, 0.0-94.0% per season). Both influenza B lineages were detected in three seasons, a single B lineage in 11, and no B strain in two. For the 11 seasons where influenza B accounted for ≥20% of positive cases, the dominant lineage was Yamagata in six and Victoria in four. In the remaining season, the two lineages co-circulated. For two seasons (2005-2006 and 2007-2008), the B lineage in the trivalent influenza vaccine did not match the dominant circulating B lineage. CONCLUSIONS: In the Czech Republic, during the 2000-2001 to 2015-2016 influenza seasons, influenza virus circulation varied considerably. Although influenza A accounted for the most cases in almost all seasons, influenza B made a substantial, sometimes dominant, contribution to influenza disease.

Correction: MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay.

[This corrects the article DOI: 10.1371/journal.pone.0151204.].

Microevolution and independent incursions as main forces shaping H5 Hemagglutinin diversity during a H5N8/H5N5 highly pathogenic avian influenza outbreak in Czech Republic in 2017.

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.

Development and evaluation of TaqMan real-time PCR assay for detection of beak and feather disease virus.

Psittacine beak and feather disease (PBFD) is one of the most significant viral diseases in psittacine birds. The aim of the presented study was to develop a highly specific and sensitive TaqMan real-time PCR assay for universal detection of beak and feather disease virus (BFDV). Primers and a hydrolysis probe were selected on the highly conserved regions belonging to the ORF1 of the BFDV genome which were identified by aligning 814 genomic sequences downloaded from the GenBank database. The evaluation of the reaction parameters suggested a reaction efficiency of 97.1%, with consistent detection of 101 virus copies/µl of nucleic acid extract. The low values of standard deviation and coefficient of variation indicate a high degree of reproducibility and repeatability. The diagnostic applicability of the assay was proven on 36 BFDV positive and 107 negative specimens of psittacine origin representing 28 species. The assay showed a 100% ability to detect distinct genetic variants of the virus. Our data suggest that the presented TaqMan real-time PCR represents a specific, sensitive and reliable assay facilitating the molecular detection of BFDV.

Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay.

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.

MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay.

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.

Large-scale nucleotide sequence alignment and sequence variability assessment to identify the evolutionarily highly conserved regions for universal screening PCR assay design: an example of influenza A virus.

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.

Local-scale diversity and between-year "frozen evolution" of avian influenza A viruses in nature.

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.

[Phylogenetic analysis and genotyping of A/H3N2 Influenza viruses isolated from patients hospitalised with influenza-like illness symptoms in the na bulovce hospital in the season 2011/2012]. / Fylogenetická analýza a genotypizace viru chripky A/H3N2 izolovaných od pacientu hospitalizovaných s príznaky ILI (Influenza-Like Illnesses) v Nemocnici Na Bulovce v sezone 2011/2012.

Influenza A virus is an important cause of acute respiratory infections (ARI). Clinical manifestations of ARI vary from mild or moderate to life-threatening conditions requiring intensive care. Given the segmented genome, a large natural reservoir of other influenza virus subtypes, and antibody selection pressure in the population, the virus is variable and genetically unstable. The phylogenetic analysis and genotyping of A/H3N2 influenza viruses isolated from patients hospitalised with influenza-like illness symptoms in the Na Bulovce Hospital in the season 2011/2012 support the assumption that the pathogenicity is a polygenic trait modifiable by the host health status and seems not to be unambiguously associated with any specific mutations.