Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia.

Phosphorylated derivatives of phosphatidylinositol (PIPs) are key membrane lipid residues involved in clathrin-mediated endocytosis (CME). CME relies on PIP species PI(4,5)P2 to mark endocytic sites at the plasma membrane (PM) associated to clathrin-coated vesicle (CCV) formation. The highly diverged parasitic protist Giardia lamblia presents disordered and static clathrin assemblies at PM invaginations, contacting specialized endocytic organelles called peripheral vacuoles (PVs). The role for clathrin assemblies in fluid phase uptake and their link to internal membranes via PIP-binding adaptors is unknown. Here we provide evidence for a robust link between clathrin assemblies and fluid-phase uptake in G. lamblia mediated by proteins carrying predicted PX, FYVE and NECAP1 PIP-binding modules. We show that chemical and genetic perturbation of PIP-residue binding and turnover elicits novel uptake and organelle-morphology phenotypes. A combination of co-immunoprecipitation and in silico analysis techniques expands the initial PIP-binding network with addition of new members. Our data indicate that, despite the partial conservation of lipid markers and protein cohorts known to play important roles in dynamic endocytic events in well-characterized model systems, the Giardia lineage presents a strikingly divergent clathrin-centered network. This includes several PIP-binding modules, often associated to domains of currently unknown function that shape and modulate fluid-phase uptake at PVs.

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.

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.

Static Clathrin Assemblies at the Peripheral Vacuole-Plasma Membrane Interface of the Parasitic Protozoan Giardia lamblia.

Giardia lamblia is a parasitic protozoan that infects a wide range of vertebrate hosts including humans. Trophozoites are non-invasive but associate tightly with the enterocyte surface of the small intestine. This narrow ecological specialization entailed extensive morphological and functional adaptations during host-parasite co-evolution, including a distinctly polarized array of endocytic organelles termed peripheral vacuoles (PVs), which are confined to the dorsal cortical region exposed to the gut lumen and are in close proximity to the plasma membrane (PM). Here, we investigated the molecular consequences of these adaptations on the Giardia endocytic machinery and membrane coat complexes. Despite the absence of canonical clathrin coated vesicles in electron microscopy, Giardia possesses conserved PV-associated clathrin heavy chain (GlCHC), dynamin-related protein (GlDRP), and assembly polypeptide complex 2 (AP2) subunits, suggesting a novel function for GlCHC and its adaptors. We found that, in contrast to GFP-tagged AP2 subunits and DRP, CHC::GFP reporters have no detectable turnover in living cells, indicating fundamental differences in recruitment to the membrane and disassembly compared to previously characterized clathrin coats. Histochemical localization in electron tomography showed that these long-lived GlCHC assemblies localized at distinctive approximations between the plasma and PV membrane. A detailed protein interactome of GlCHC revealed all of the conserved factors in addition to novel or highly diverged proteins, including a putative clathrin light chain and lipid-binding proteins. Taken together, our data provide strong evidence for giardial CHC as a component of highly stable assemblies at PV-PM junctions that likely have a central role in organizing continuities between the PM and PV membranes for controlled sampling of the fluid environment. This suggests a novel function for CHC in Giardia and the extent of molecular remodeling of endocytosis in this species.

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.

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.

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.

Canine Distemper Virus in Wild Carnivore Populations from the Czech Republic (2012-2020): Occurrence, Geographical Distribution, and Phylogenetic Analysis.

Canine distemper is a highly contagious viral disease in carnivores and represents a serious threat for both wild and domestic animals. The aim of our study was to monitor the occurrence of the canine distemper virus in wildlife from the Czech Republic, reveal the H gene heterogeneity in positive samples and perform subsequent phylogenetic analysis. In total, 412 wild animals of 10 species were included in the study: 219 red foxes (Vulpes vulpes), 79 European badgers (Meles meles), 47 European otters (Lutra lutra), 40 stone martens (Martes foina), 10 pine martens (M. martes), 7 raccoons (Procyon lotor), 5 undetermined martens (Martes sp.), 2 wolves (Canis lupus), 1 European polecat (Mustela putorius), 1 free-ranging ferret (Mustela putorius furo), and 1 free-ranging American mink (Neovison vison). Most animals were found dead or were killed by hunters during hunting seasons in the years 2012-2020 and came from all 14 regions of the Czech Republic. In the animals that were hunted, symptoms such as apathy, loss of shyness or disorientation were reported. Canine distemper virus (CDV) was detected by real-time RT-PCR in the tissues of 74 (18%) of the animals, including 62 (28%) red foxes, 4 (10%) stone martens, 3 (43%) raccoons, 2 (20%) pine martens, 2 (2.5%) European badgers and 1 (20%) undetermined marten. There was a statistical difference in positivity among animal species (p < 0.0001), regions (p = 0.0057), and the years of sampling (p = 0.0005). To determine the genetic characteristics of circulating variants of CDV in wildlife, 23 of 74 CDV variants were partially sequenced. Phylogenetic analysis showed that 21 variants belonged to the European lineage and two strains belonged to the European-Wildlife lineage. This study provides the first comprehensive overview of the prevalence and spatial distribution of CDV in wildlife in the Czech Republic, including molecular phylogenetic analysis of currently circulating CDV lineages.

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.

Disentangling the role of poultry farms and wild birds in the spread of highly pathogenic avian influenza virus in Europe.

In winter 2016-7, Europe was severely hit by an unprecedented epidemic of highly pathogenic avian influenza viruses (HPAIVs), causing a significant impact on animal health, wildlife conservation, and livestock economic sustainability. By applying phylodynamic tools to virus sequences collected during the epidemic, we investigated when the first infections occurred, how many infections were unreported, which factors influenced virus spread, and how many spillover events occurred. HPAIV was likely introduced into poultry farms during the autumn, in line with the timing of wild birds' migration. In Germany, Hungary, and Poland, the epidemic was dominated by farm-to-farm transmission, showing that understanding of how farms are connected would greatly help control efforts. In the Czech Republic, the epidemic was dominated by wild bird-to-farm transmission, implying that more sustainable prevention strategies should be developed to reduce HPAIV exposure from wild birds. Inferred transmission parameters will be useful to parameterize predictive models of HPAIV spread. None of the predictors related to live poultry trade, poultry census, and geographic proximity were identified as supportive predictors of HPAIV spread between farms across borders. These results are crucial to better understand HPAIV transmission dynamics at the domestic-wildlife interface with the view to reduce the impact of future epidemics.

Wild Small Mammals and Ticks in Zoos-Reservoir of Agents with Zoonotic Potential?

Wild small mammals and ticks play an important role in maintaining and spreading zoonoses in nature, as well as in captive animals. The aim of this study was to monitor selected agents with zoonotic potential in their reservoirs and vectors in a zoo, and to draw attention to the risk of possible contact with these pathogens. In total, 117 wild small mammals (rodents) and 166 ticks were collected in the area of Brno Zoo. Antibodies to the bacteria Coxiella burnetii, Francisella tularensis, and Borrelia burgdorferi s.l. were detected by a modified enzyme-linked immunosorbent assay in 19% (19/99), 4% (4/99), and 15% (15/99) of rodents, respectively. Antibodies to Leptospira spp. bacteria were detected by the microscopic agglutination test in 6% (4/63) of rodents. Coinfection (antibodies to more than two agents) were proved in 14.5% (15/97) of animals. The prevalence of C. burnetii statistically differed according to the years of trapping (p = 0.0241). The DNAs of B. burgdorferi s.l., Rickettsia sp., and Anaplasma phagocytophilum were detected by PCR in 16%, 6%, and 1% of ticks, respectively, without coinfection and without effect of life stage and sex of ticks on positivity. Sequencing showed homology with R. helvetica and A. phagocytophilum in four and one positive samples, respectively. The results of our study show that wild small mammals and ticks in a zoo could serve as reservoirs and vectors of infectious agents with zoonotic potential and thus present a risk of infection to zoo animals and also to keepers and visitors to a zoo.

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.

Development and evaluation of a one-step real-time RT-PCR assay for universal detection of influenza A viruses from avian and mammal species.

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.

Molecular and phylogenetic analysis of the H5N1 avian influenza virus caused the first highly pathogenic avian influenza outbreak in poultry in the Czech Republic in 2007.

On 19th July 2007 re-occurrence of the H5N1 highly pathogenic avian influenza (HPAI) virus was noticed in Europe. The index strain of this novel H5N1 lineage was identified in the Czech Republic where it caused historically the first HPAI outbreak in commercial poultry. In the present study we performed molecular and phylogenetic analysis of the index strain of the re-emerging H5N1 virus lineage along with the Czech and the Slovak H5N1 strains collected in 2006 and established the evolutionary relationships to additional viruses circulated in Europe in 2005-2006. Our analysis revealed that the Czech and the Slovak H5N1 viruses collected during 2006 were separated into two sub-clades 2.2.1 and 2.2.2, which predominated in Europe during 2005-2006. On the contrary the newly emerged H5N1 viruses belonged to a clearly distinguishable sub-clade 2.2.3. Within the sub-clade 2.2.3 the Czech H5N1 strains showed the closest relationships to the simultaneously circulated viruses from Germany, Romania and Russia (Krasnodar) in 2007 and were further clustered with the viruses from Afghanistan and Mongolia circulated in 2006. The origin of the Czech 2007 H5N1 HPAI strains was also discussed.

Roles of Phosphoinositides and Their binding Proteins in Parasitic Protozoa.

Phosphoinositides (or phosphatidylinositol phosphates, PIPs) are low-abundance membrane phospholipids that act, in conjunction with their binding partners, as important constitutive signals defining biochemical organelle identity as well as membrane trafficking and signal transduction at eukaryotic cellular membranes. In this review, we present roles for PIP residues and PIP-binding proteins in endocytosis and autophagy in protist parasites such as Trypanosoma brucei, Toxoplasma gondii, Plasmodium falciparum, Entamoeba histolytica, and Giardia lamblia. Molecular parasitologists with an interest in comparative cell and molecular biology of membrane trafficking in protist lineages beyond the phylum Apicomplexa, along with cell and molecular biologists generally interested in the diversification of membrane trafficking in eukaryotes, will hopefully find this review to be a useful resource.

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.

Serologic Survey of Selected Viral Pathogens in Free-Ranging Eurasian Brown Bears ( Ursus arctos arctos) from Slovakia.

We tested sera of 24 free-ranging European brown bears ( Ursus arctos) from six regions of Slovakia for antibodies to 10 viral agents. We tested sera by an indirect fluorescence antibody test for antibodies to canine distemper virus (CDV), canine coronavirus (CCV), canine parvovirus type 2 (CPV-2), canine adenovirus, canine parainfluenza virus type 2 (CPIV-2), and canine herpesvirus type 1 (CHV-1). We used an enzyme-linked immunosorbent assay for detection of antibodies to hepatitis E virus, bluetongue virus, West Nile virus (WNV), and Aujeszky's disease virus (ADV). We detected antibodies to CDV, CHV-1, CPV-2, CPIV-2, CCV, WNV, and ADV in seven (29%), three (12%), two (8%), two (8%), one (4%), one (4%), and one (4%) bear, respectively. Evidence of exposure of free-ranging European brown bears to CCV and ADV has not been reported.

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.].