Rescue and in vitro characterization of a divergent TBEV-Eu strain from the Netherlands.

Tick-borne encephalitis virus (TBEV) may cause tick-borne encephalitis (TBE), a potential life-threatening infection of the central nervous system in humans. Phylogenetically, TBEVs can be subdivided into three main subtypes, which differ in endemic region and pathogenic potential. In 2016, TBEV was first detected in the Netherlands. One of two detected strains, referred to as Salland, belonged to the TBEV-Eu subtype, yet diverged ≥ 2% on amino acid level from other members of this subtype. Here, we report the successful rescue of this strain using infectious subgenomic amplicons and its subsequent in vitro characterization by comparison to two well-characterized TBEV-Eu strains; Neudoerfl and Hypr. In the human alveolar epithelial cell line A549, growth kinetics of Salland were comparable to the high pathogenicity TBEV-Eu strain Hypr, and both strains grew considerably faster than the mildly pathogenic strain Neudoerfl. In the human neuroblastoma cell line SK-N-SH, Salland replicated faster and to higher infectious titers than both reference strains. All three TBEV strains infected primary human monocyte-derived dendritic cells to a similar extent and interacted with the type I interferon system in a similar manner. The current study serves as the first in vitro characterization of the novel, divergent TBEV-Eu strain Salland.

Distinguishing Echinococcus granulosus sensu stricto genotypes G1 and G3 with confidence: A practical guide.

Cystic echinococcosis (CE), a zoonotic disease caused by tapeworms of the species complex Echinococcus granulosus sensu lato, represents a substantial global health and economic burden. Within this complex, E. granulosus sensu stricto (genotypes G1 and G3) is the most frequent causative agent of human CE. Currently, there is no fully reliable method for assigning samples to genotypes G1 and G3, as the commonly used mitochondrial cox1 and nad1 genes are not sufficiently consistent for the identification and differentiation of these genotypes. Thus, a new genetic assay is required for the accurate assignment of G1 and G3. Here we use a large dataset of near-complete mtDNA sequences (n = 303) to reveal the extent of genetic variation of G1 and G3 on a broad geographical scale and to identify reliable informative positions for G1 and G3. Based on extensive sampling and sequencing data, we developed a new method, that is simple and cost-effective, to designate samples to genotypes G1 and G3. We found that the nad5 is the best gene in mtDNA to differentiate between G1 and G3, and developed new primers for the analysis. Our results also highlight problems related to the commonly used cox1 and nad1. To guarantee consistent identification of G1 and G3, we suggest using the sequencing of the nad5 gene region (680 bp). This region contains six informative positions within a relatively short fragment of the mtDNA, allowing the differentiation of G1 and G3 with confidence. Our method offers clear advantages over the previous ones, providing a significantly more consistent means to distinguish G1 and G3 than the commonly used cox1 and nad1.

Global phylogeography and genetic diversity of the zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1.

Echinococcus granulosus sensu stricto (s.s.) is the major cause of human cystic echinococcosis worldwide and is listed among the most severe parasitic diseases of humans. To date, numerous studies have investigated the genetic diversity and population structure of E. granulosus s.s. in various geographic regions. However, there has been no global study. Recently, using mitochondrial DNA, it was shown that E. granulosus s.s. G1 and G3 are distinct genotypes, but a larger dataset is required to confirm the distinction of these genotypes. The objectives of this study were to: (i) investigate the distinction of genotypes G1 and G3 using a large global dataset; and (ii) analyse the genetic diversity and phylogeography of genotype G1 on a global scale using near-complete mitogenome sequences. For this study, 222 globally distributed E. granulosus s.s. samples were used, of which 212 belonged to genotype G1 and 10 to G3. Using a total sequence length of 11,682 bp, we inferred phylogenetic networks for three datasets: E. granulosus s.s. (n = 222), G1 (n = 212) and human G1 samples (n = 41). In addition, the Bayesian phylogenetic and phylogeographic analyses were performed. The latter yielded several strongly supported diffusion routes of genotype G1 originating from Turkey, Tunisia and Argentina. We conclude that: (i) using a considerably larger dataset than employed previously, E. granulosus s.s. G1 and G3 are indeed distinct mitochondrial genotypes; (ii) the genetic diversity of E. granulosus s.s. G1 is high globally, with lower values in South America; and (iii) the complex phylogeographic patterns emerging from the phylogenetic and geographic analyses suggest that the current distribution of genotype G1 has been shaped by intensive animal trade.

High-resolution phylogeography of zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1 with an emphasis on its distribution in Turkey, Italy and Spain.

Echinococcus granulosus is the causative agent of cystic echinococcosis. The disease is a significant global public health concern and human infections are most commonly associated with E. granulosus sensu stricto (s. s.) genotype G1. The objectives of this study were to: (i) analyse the genetic variation and phylogeography of E. granulosus s. s. G1 in part of its main distribution range in Europe using 8274 bp of mtDNA; (ii) compare the results with those derived from previously used shorter mtDNA sequences and highlight the major differences. We sequenced a total of 91 E. granulosus s. s. G1 isolates from six different intermediate host species, including humans. The isolates originated from seven countries representing primarily Turkey, Italy and Spain. Few samples were also from Albania, Greece, Romania and from a patient originating from Algeria, but diagnosed in Finland. The analysed 91 sequences were divided into 83 haplotypes, revealing complex phylogeography and high genetic variation of E. granulosus s. s. G1 in Europe, particularly in the high-diversity domestication centre of western Asia. Comparisons with shorter mtDNA datasets revealed that 8274 bp sequences provided significantly higher phylogenetic resolution and thus more power to reveal the genetic relations between different haplotypes.

Screening of bat faeces for arthropod-borne apicomplexan protozoa: Babesia canis and Besnoitia besnoiti-like sequences from Chiroptera.

BACKGROUND: Bats are among the most eco-epidemiologically important mammals, owing to their presence in human settlements and animal keeping facilities. Roosting of bats in buildings may bring pathogens of veterinary-medical importance into the environment of domestic animals and humans. In this context bats have long been studied as carriers of various pathogen groups. However, despite their close association with arthropods (both in their food and as their ectoparasites), only a few molecular surveys have been published on their role as carriers of vector-borne protozoa. The aim of the present study was to compensate for this scarcity of information. FINDINGS: Altogether 221 (mostly individual) bat faecal samples were collected in Hungary and the Netherlands. The DNA was extracted, and analysed with PCR and sequencing for the presence of arthropod-borne apicomplexan protozoa. Babesia canis canis (with 99-100% homology) was identified in five samples, all from Hungary. Because it was excluded with an Ixodidae-specific PCR that the relevant bats consumed ticks, these sequences derive either from insect carriers of Ba. canis, or from the infection of bats. In one bat faecal sample from the Netherlands a sequence having the highest (99%) homology to Besnoitia besnoiti was amplified. CONCLUSIONS: These findings suggest that some aspects of the epidemiology of canine babesiosis are underestimated or unknown, i.e. the potential role of insect-borne mechanical transmission and/or the susceptibility of bats to Ba. canis. In addition, bats need to be added to future studies in the quest for the final host of Be. besnoiti.

Ability to cause erythema migrans differs between Borrelia burgdorferi sensu lato isolates.

BACKGROUND: Lyme borreliosis is a tick-borne disease caused by Borrelia burgdorferi sensu lato. The variety of characteristic and non-specific clinical manifestations is partially explained by its genetic diversity. We investigated the ability of B. burgdorferi sl isolates to cause erythema migrans. METHODS: The genetic constellation of isolates from ticks was compared to isolates found in erythema migrans. PCR and sequence analysis was performed on the plasmid-encoded ospC and the chromosomal 5S-23S rDNA spacer region (IGS). RESULTS: Seven different B. burgdorferi sl genospecies were identified in 152 borrelia isolates from ticks and erythema migrans biopsies. B afzelii (51%) and B. garinii (27%) were the most common in ticks. From the 44 sequences obtained from erythema migrans samples 42 were B. afzelii, one B. garinii and one B. bavariensis. Significant associations with erythema migrans formation were found for four IGS and two ospC types. Five from 45 ospC types were associated with more than one genospecies. CONCLUSIONS: B. burgdorferi sl isolates differ in their propensity to cause erythema migrans. These differences were also found within genospecies. In other words, although B. afzelii was mostly associated with erythema migrans, some B. afzelii isolates had a low ability to cause erythema migrans. Our data further support the occurrence of plasmid exchange between borrelia genospecies under natural conditions.

The lifetime of UDP-galactose:ceramide galactosyltransferase is controlled by a distinct endoplasmic reticulum-associated degradation (ERAD) regulated by sigma-1 receptor chaperones.

The glycosphingolipid biosynthesis is initiated by monoglycosylation of ceramides, the action of which is catalyzed either by UDP-glucose:ceramide glucosyltransferase or by UDP-galactose:ceramide galactosyltransferase (CGalT). CGalT is expressed predominantly at the endoplasmic reticulum (ER) of oligodendrocytes and is responsible for synthesizing galactosylceramides (GalCer) that play an important role in regulation of axon conductance. However, despite the importance of ceramide monoglycosylation enzymes in a spectrum of cellular functions, the mechanism that fine tunes activities of those enzymes is largely unknown. In the present study, we demonstrated that the sigma-1 receptor (Sig-1R) chaperone, the mammalian homologue of a yeast C8-C7 sterol isomerase, controls the protein level and activity of the CGalT enzyme via a distinct ER-associated degradation system involving Insig. The Sig-1R forms a complex with Insig via its transmembrane domain partly in a sterol-dependent manner and associates with CGalT at the ER. The knockdown of Sig-1Rs dramatically prolonged the lifetime of CGalT without affecting the trimming of N-linked oligosaccharides at CGalT. The increased lifetime leads to the up-regulation of CGalT protein as well as elevated enzymatic activity in CHO cells stably expressing CGalT. Knockdown of Sig-1Rs also decreased CGalT degradation endogenously expressed in D6P2T-schwannoma cells. Our data suggest that Sig-1Rs negatively regulate the activity of GalCer synthesis under physiological conditions by enhancing the degradation of CGalT through regulation of the dynamics of Insig in the lipid-activated ER-associated degradation system. The GalCer synthesis may thus be influenced by sterols at the ER.

Population-based analyses of Giardia duodenalis is consistent with the clonal assemblage structure.

BACKGROUND: Giardia duodenalis is a common protozoan parasite of humans and animals. Genetic characterization of single loci indicates the existence of eight groups called assemblages, which differ in their host distribution. Molecular analyses challenged the idea that G. duodenalis is a strictly clonal diplomonad by providing evidence of recombination within and between assemblages. Particularly, inter-assemblage recombination events would complicate the interpretation of multi-locus genotyping data from field isolates: where is a host infected with multiple Giardia genotypes or with a single, recombined Giardia genotype. METHODS: Population genetic analyses on the single and multiple-locus level on an extensive dataset of G. duodenalis isolates from humans and animals were performed. RESULTS: Our analyses indicate that recombination between isolates from different assemblages are apparently very rare or absent in the natural population of Giardia duodenalis. At the multi-locus level, our statistical analyses are more congruent with clonal reproduction and can equally well be explained with the presence of multiple G. duodenalis genotypes within one field isolate. CONCLUSIONS: We conclude that recombination between G. duodenalis assemblages is either very rare or absent. Recombination between genotypes from the same assemblage and genetic exchange between the nuclei of a single cyst needs further investigation.

Hyperacidification of trans-Golgi network and endo/lysosomes in melanocytes by glucosylceramide-dependent V-ATPase activity.

Sphingolipids are considered to play a key role in protein sorting and membrane trafficking. In melanocytic cells, sorting of lysosomal and melanosomal proteins requires the sphingolipid glucosylceramide (GlcCer). This sorting information is located in the lumenal domain of melanosomal proteins. We found that two processes dependent on lumenal pH, protein sialylation and lysosomal acid lipase (LAL) activity were aberrant in GM95 melanocyte cells, which do not produce glycosphingolipids. Using fluorescence lifetime imaging microscopy (FLIM), we found that the lumenal pH in the trans-Golgi network and lysosomes of wild-type melanocyte MEB4 cells are >1 pH unit lower than GM95 cells and fibroblasts. In addition to the lower pH found in vivo, the in vitro activity of the proton pump, the vacuolar-type H(+) -translocating ATPase (V-ATPase), was twofold higher in MEB4 compared to GM95 cells. The apparent K(i) for inhibition of the V-ATPase by concanamycin A and archazolid A, which share a common binding site on the c-ring, was lower in glycosphingolipid-deficient GM95 cells. No difference between the MEB4 and GM95 cells was found for the V-ATPase inhibitors apicularen A and salicylihalimide. We conclude that hyperacidification in MEB4 cells requires glycosphingolipids and propose that low pH is necessary for protein sorting and melanosome biogenesis. Furthermore, we suggest that glycosphingolipids are indirectly involved in protein sorting and melanosome biogenesis by stimulating the proton pump, possibly through binding of GlcCer. These experiments establish, for the first time, a link between pH, glycosphingolipids and melanosome biogenesis in melanocytic MEB4 cells, to suggest a role for glycosphingolipids in hyperacidification in melanocytes.

Prevalence and molecular typing of Giardia spp. in captive mammals at the zoo of Zagreb, Croatia.

A total of 131 faecal samples from 57 mammalian species housed at the zoo of Zagreb, Croatia, were tested for the presence of Giardia spp. cysts using epifluorescence microscopy. The overall prevalence (29%) was high, yet all animals were asymptomatic at the time of sampling. Positive samples were characterized by PCR and sequence analysis of both conserved and variable loci, for the identification of Giardia species and G. duodenalis assemblages and genotypes. Assemblages A and C were identified in Artiodactyla, assemblage B in Primates, Rodentia and Hyracoidea, and assemblages A, B, C and D, as well as Giardia microti, in Carnivora. Genotyping at the ITS1-5.8S-ITS2 region, at the triose phosphate isomerase, glutamate dehydrogenase and beta-giardin genes revealed extensive polymorphisms, particularly among assemblage B isolates. A phylogenetic analysis of concatenated sequences showed that isolates from captive mammals housed at the zoo are genetically different from isolates of human and domestic animal origin. This is the first survey in a zoological garden to include a molecular characterization of the parasite, and provides novel sequence data of G. duodenalis from many previously uncharacterized hosts.

A large survey of Croatian wild mammals for Giardia duodenalis reveals a low prevalence and limited zoonotic potential.

Wild mammals are considered an important source of potentially zoonotic Giardia duodenalis parasites, yet surprisingly little information is available on the actual prevalence and the genetic identity of the species they harbor. A large survey was conducted in Croatia by collecting 832 fecal samples from red deer (Cervus elaphus, n = 374), roe deer (Capreolus capreolus, n = 21), wild boars (Sus scrofa, n = 144), foxes (Vulpes vulpes, n = 66), bears (Ursus arctos, n = 19), wolves (Canis lupus, n = 127), jackals (Canis aureus, n = 8), and hares (Lepus europeus, n = 73). Fecal samples were tested for the presence of Giardia cysts using fluorescent microscopy. The observed prevalence ranged from low (1% in red deer, 1.7% in wild boars, and 4.5% in foxes) to moderate (10% in wolves and 12.5% in jackals) to high (24% in roe deer). No cysts were observed in bears and hares. Polymerase chain reaction was performed on microscopically positive samples to amplify fragments of the small subunit ribosomal gene, the ribosomal 5.8S gene and the two flanking internal transcribed sequences, and the triose phosphate isomerase gene. Sequence analysis showed a predominance of G. duodenalis assemblage A in both ruminants (genotypes A1 and A3) and carnivores (genotype A1). G. duodenalis assemblages B, C, and D, as well as Giardia microti, were also detected in this study. This is the first molecular description of the parasite from the red deer, the wolf, and the jackal. The data point to a minor role of wild mammals as reservoirs of zoonotic assemblages of G. duodenalis, albeit cycling between sylvatic and domestic animals is possible.

Giardia duodenalis: genetic recombination and its implications for taxonomy and molecular epidemiology.

Traditionally, species within the Giardia genus have been considered as eukaryotic organisms that show an absence of sexual reproduction in their simple life cycles. This apparent lack of sex has been challenged by a number of studies that have demonstrated (i) the presence in the Giardia duodenalis genome of true homologs of genes specifically involved in meiosis in other eukaryotes, and their stage-specific expression; (ii) the exchange of genetic material in different chromosomal regions among human isolates of the parasite; (iii) the fusion between cyst nuclei (karyogamy) and the transfer of genetic material (episomal plasmids) between them. These results are pivotal for the existence of sexual recombination. However, many details of the process remain elusive, and experimental data are still scarce. This review summarizes the experimental approaches and the results obtained, and discusses the implications of recombination from the standpoint of the taxonomy and molecular epidemiology of this widespread pathogen.

Identification of zoonotic genotypes of Giardia duodenalis.

Giardia duodenalis, originally regarded as a commensal organism, is the etiologic agent of giardiasis, a gastrointestinal disease of humans and animals. Giardiasis causes major public and veterinary health concerns worldwide. Transmission is either direct, through the faecal-oral route, or indirect, through ingestion of contaminated water or food. Genetic characterization of G. duodenalis isolates has revealed the existence of seven groups (assemblages A to G) which differ in their host distribution. Assemblages A and B are found in humans and in many other mammals, but the role of animals in the epidemiology of human infection is still unclear, despite the fact that the zoonotic potential of Giardia was recognised by the WHO some 30 years ago. Here, we performed an extensive genetic characterization of 978 human and 1440 animal isolates, which together comprise 3886 sequences from 4 genetic loci. The data were assembled into a molecular epidemiological database developed by a European network of public and veterinary health Institutions. Genotyping was performed at different levels of resolution (single and multiple loci on the same dataset). The zoonotic potential of both assemblages A and B is evident when studied at the level of assemblages, sub-assemblages, and even at each single locus. However, when genotypes are defined using a multi-locus sequence typing scheme, only 2 multi-locus genotypes (MLG) of assemblage A and none of assemblage B appear to have a zoonotic potential. Surprisingly, mixtures of genotypes in individual isolates were repeatedly observed. Possible explanations are the uptake of genetically different Giardia cysts by a host, or subsequent infection of an already infected host, likely without overt symptoms, with a different Giardia species, which may cause disease. Other explanations for mixed genotypes, particularly for assemblage B, are substantial allelic sequence heterogeneity and/or genetic recombination. Although the zoonotic potential of G. duodenalis is evident, evidence on the contribution and frequency is (still) lacking. This newly developed molecular database has the potential to tackle intricate epidemiological questions concerning protozoan diseases.

[Reintroduction of E. granulosus by import of cows in the Netherlands]. / Herintroductie van echinococcose via runderimport in Nederland.

Since East European countries joined the EU, the import of both dairy and beef cows from these countries increased considerably. Based on the identification and registration system it turned out that in the period from May until December 2007 about 200 cows per month were imported from Romania. These animals were either slaughtered immediately or in autumn. In autumn, cysts were noticed both in slaughtered cows during meat inspection and in deceased animals (originated from Romania) during postmortem investigation performed by the Animal Health Service. Because cysts were strongly reminiscent of Echinococcus granulosus hydatid cysts, samples were sent to the authorized laboratory (National Reference Laboratory of Parasitology), where the reintroduction of this potentially zoonotic parasitic infection has been confirmed. The risks of reintroduction of E. granulosus in the Netherlands are described.

Glycolipid-dependent sorting of melanosomal from lysosomal membrane proteins by lumenal determinants.

Melanosomes are lysosome-related organelles that coexist with lysosomes in mammalian pigment cells. Melanosomal and lysosomal membrane proteins share similar sorting signals in their cytoplasmic tail, raising the question how they are segregated. We show that in control melanocytes, the melanosomal enzymes tyrosinase-related protein 1 (Tyrp1) and tyrosinase follow an intracellular Golgi to melanosome pathway, whereas in the absence of glycosphingolipids, they are observed to pass over the cell surface. Unexpectedly, the lysosome-associated membrane protein 1 (LAMP-1) and 2 behaved exactly opposite: they were found to travel through the cell surface in control melanocytes but followed an intracellular pathway in the absence of glycosphingolipids. Chimeric proteins having the cytoplasmic tail of Tyrp1 or tyrosinase were transported like lysosomal proteins, whereas a LAMP-1 construct containing the lumenal domain of Tyrp1 localized to melanosomes. In conclusion, the lumenal domain contains sorting information that guides Tyrp1 and probably tyrosinase to melanosomes by an intracellular route that excludes lysosomal proteins and requires glucosylceramide.

Gangliosides play an important role in the organization of CD82-enriched microdomains.

Four-transmembrane-domain proteins of the tetraspanin superfamily are the organizers of specific microdomains at the membrane [TERMs (tetraspanin-enriched microdomains)] that incorporate various transmembrane receptors and modulate their activities. The structural aspects of the organization of TERM are poorly understood. In the present study, we investigated the role of gangliosides in the assembly and stability of TERM. We demonstrated that inhibition of the glycosphingolipid biosynthetic pathway with specific inhibitors of glucosylceramide synthase [NB-DGJ (N-butyldeoxygalactonojirimycin) and PPMP (D-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol.HCl)] resulted in specific weakening of the interactions involving tetraspanin CD82. Furthermore, ectopic expression of the plasma-membrane-bound sialidase Neu3 in mammary epithelial cells also affected stability of the complexes containing CD82: its association with tetraspanin CD151 was decreased, but the association with EGFR [EGF (epidermal growth factor) receptor] was enhanced. The destabilization of the CD82-containing complexes upon ganglioside depletion correlated with the re-distribution of the proteins within plasma membrane. Importantly, depletion of gangliosides affected EGF-induced signalling only in the presence of CD82. Taken together, our results provide strong evidence that gangliosides play an important role in supporting the integrity of CD82-enriched microdomains. Furthermore, these results demonstrate that the association between different tetraspanins in TERM is controlled by distinct mechanisms and identify Neu3 as a first physiological regulator of the integrity of these microdomains.

Lipoprotein particles are required for Hedgehog and Wingless signalling.

Wnt and Hedgehog family proteins are secreted signalling molecules (morphogens) that act at both long and short range to control growth and patterning during development. Both proteins are covalently modified by lipid, and the mechanism by which such hydrophobic molecules might spread over long distances is unknown. Here we show that Wingless, Hedgehog and glycophosphatidylinositol-linked proteins copurify with lipoprotein particles, and co-localize with them in the developing wing epithelium of Drosophila. In larvae with reduced lipoprotein levels, Hedgehog accumulates near its site of production, and fails to signal over its normal range. Similarly, the range of Wingless signalling is narrowed. We propose a novel function for lipoprotein particles, in which they act as vehicles for the movement of lipid-linked morphogens and glycophosphatidylinositol-linked proteins.

Membrane lipids and vesicular traffic.

Lipids were long considered to be passive passengers of carrier vesicles with the single role of sealing the transport container. We now know that specific phospholipids are required for efficient fusion, while others facilitate budding and fission. Moreover, the various polyphosphoinositides assist in the recruitment from the cytosol of proteins of the transport machinery. Finally, the segregation of membrane lipids into different fluid phases appears to serve as a 'lipid raft' mechanism for protein sorting at various stages of the secretory and endocytic pathways. The current challenge is to understand how proteins control the metabolism and subcellular localization, and thereby the activity, of the various lipids.