Every year, tick-borne encephalitis virus (TBEV) causes severe central nervous system infection in 10 000 to 15 000 people in Europe and Asia. TBEV is maintained in the environment by an enzootic cycle that requires a tick vector and a vertebrate host, and the adaptation of TBEV to vertebrate and invertebrate environments is essential for TBEV persistence in nature. This adaptation is facilitated by the error-prone nature of the virus's RNA-dependent RNA polymerase, which generates genetically distinct virus variants called quasispecies. TBEV shows a focal geographical distribution pattern where each focus represents a TBEV hotspot. Here, we sequenced and characterized two TBEV genomes, JP-296 and JP-554, from questing Ixodes ricinus ticks at a TBEV focus in central Sweden. Phylogenetic analysis showed geographical clustering among the newly sequenced strains and three previously sequenced Scandinavian strains, Toro-2003, Saringe-2009 and Mandal-2009, which originated from the same ancestor. Among these five Scandinavian TBEV strains, only Mandal-2009 showed a large deletion within the 3' non-coding region (NCR), similar to the highly virulent TBEV strain Hypr. Deep sequencing of JP-296, JP-554 and Mandal-2009 revealed significantly high quasispecies diversity for JP-296 and JP-554, with intact 3' NCRs, compared to the low diversity in Mandal-2009, with a truncated 3' NCR. Single-nucleotide polymorphism analysis showed that 40% of the single-nucleotide polymorphisms were common between quasispecies populations of JP-296 and JP-554, indicating a putative mechanism for how TBEV persists and is maintained within its natural foci.

The hypothesis of wide spread reticulate evolution in Tick-Borne Encephalitis virus (TBEV) has recently gained momentum with several publications describing past recombination events involving various TBEV clades. Despite a large body of work, no consensus has yet emerged on TBEV evolutionary dynamics. Understanding the occurrence and frequency of recombination in TBEV bears significant impact on epidemiology, evolution, and vaccination with live vaccines. In this study, we investigated the possibility of detecting recombination events in TBEV by simulating recombinations at several locations on the virus' phylogenetic tree and for different lengths of recombining fragments. We derived estimations of rates of true and false positive for the detection of past recombination events for seven recombination detection algorithms. Our analytical framework can be applied to any investigation dealing with the difficult task of distinguishing genuine recombination signal from background noise. Our results suggest that the problem of false positives associated with low detection P-values in TBEV, is more insidious than generally acknowledged. We reappraised the recombination signals present in the empirical data, and showed that reliable signals could only be obtained in a few cases when highly genetically divergent strains were involved, whereas false positives were common among genetically similar strains. We thus conclude that recombination among wild-type TBEV strains may occur, which has potential implications for vaccination with live vaccines, but that these events are surprisingly rare.

The tick-borne encephalitis virus (TBEV) is a flavivirus transmitted to humans, usually via tick bites. The virus causes tick-borne encephalitis (TBE) in humans, and symptoms range from mild flu-like symptoms to severe and long-lasting sequelae, including permanent brain damage. It has been suggested that within the population of viruses transmitted to the mammalian host, quasispecies with neurotropic properties might become dominant in the host resulting in neurological symptoms. We previously demonstrated the existence of TBEV variants with variable poly(A) tracts within a single blood-fed tick. To characterize the role of the poly(A) tract in TBEV replication and virulence, we generated infectious clones of Torö-2003 with the wild-type (A)3C(A)6 sequence (Torö-6A) or with a modified (A)3C(A)38 sequence (Torö-38A). Torö-38A replicated poorly compared to Torö-6A in cell culture, but Torö-38A was more virulent than Torö-6A in a mouse model of TBE. Next-generation sequencing of TBEV genomes after passaging in cell culture and/or mouse brain revealed mutations in specific genomic regions and the presence of quasispecies that might contribute to the observed differences in virulence. These data suggest a role for quasispecies development within the poly(A) tract as a virulence determinant for TBEV in mice.

The increased distribution of the tick-borne encephalitis virus (TBEV) in Scandinavia highlights the importance of characterizing novel sequences within the natural foci. In this study, two TBEV strains: the Norwegian Mandal 2009 (questing nymphs pool) and the Swedish Saringe 2009 (blood-fed nymph) were sequenced and phylogenetically characterized. Interestingly, the sequence of Mandal 2009 revealed the shorter form of the TBEV genome, similar to the highly virulent Hypr strain, within the 3' non-coding region (3'NCR). A different genomic structure was found in the 3'NCR of Saringe 2009, as in-depth analysis demonstrated TBEV variants with different lengths within the poly(A) tract. This shows that TBEV quasispecies exists in nature and indicates a putative shift in the quasispecies pool when the virus switches between invertebrate and vertebrate environments. This prompted us to further sequence and analyze the 3'NCRs of additional Scandinavian TBEV strains and control strains, Hypr and Neudoerfl. Toro 2003 and Habo 2011 contained mainly a short (A)3C(A)6 poly(A) tract. A similar pattern was observed for the human TBEV isolates 1993/783 and 1991/4944; however, one clone of 1991/4944 contained an (A)3C(A)11 poly(A) sequence, demonstrating that quasispecies with longer poly(A) could be present in human isolates. Neudoerfl has previously been reported to contain a poly(A) region, but to our surprise the re-sequenced genome contained two major quasispecies variants, both lacking the poly(A) tract. We speculate that the observed differences are important factors for the understanding of virulence, spread, and control of the TBEV.

The re-emergence and modernization of traditional goat-cheese production in Jamtland led to the articulation of a localized agri-food system that represents the frontline of the return and reinforcement of local food in Sweden. Already in the 1970s, some initiatives were undertaken to formalize the productive activities of this branch and to improve the product quality. The most important project was the articulation of a cooperative that, unlike all other Swedish cooperatives, engaged its members in the development of a joint trademark, development of a standardized assortment, common marketing efforts and finding creative solutions for infrastructure problems. Despite the overall success, we also found some downsides. Producing goat cheese requires that at least two people are involved, because the workload often leads to body injuries and illness for people working alone. By studying the institutional frameworks, rules and regulations, the economic function and entrepr! neurial dynamics, and the dynamics of knowledge and competences, the article highlights how and why farm dairies in Jamtland became reinforced and modernized. This grasps both the actions of individual economic agents and their interaction with their environment. A special emphasis was put on the role of regional authorities in this process. Even though many obstacles have been removed and the trade has found successful ways to solve strategic issues concerning product development and marketing, there are still important structural shortcomings that might decrease the profitability and endanger the future development of the trade. There is a lack of experience and infrastructure to solve more complex problems like animal healthand the potential risks related to the consumption of unpasteurized cheese and the increasing incidence of Tick-Borne Encephalitis (TBE).

Patients with IgE antibodies against the carbohydrate epitope galactose--1,3-galactose (-Gal) have reported severe allergic reactions after consumption of red meat. Investigations have revealed associations between IgE to -Gal and tick bites. We provide the first direct evidence that -Gal is present within ticks thus potentially explaining the relationship between tick exposure and sensitization to -Gal, with development of red meat allergy as a secondary phenomena. Serum from Swedish patients with delayed severe reactions to red meat was included in the study. A dose-dependent inhibition of IgE responses to -Gal by the tick Ixodesricinus is demonstrated. Furthermore, using cryostat-cut sections of I.ricinus, we show that both a monoclonal and a polyclonal antibody against -Gal stains the gastrointestinal tract of the tick. The same pattern is seen when staining with patient sera IgE positive to -Gal. These results confirm that the -Gal epitope is present in I.ricinus and imply host exposure to -Gal during a tick bite. This provides further evidence that tick bites are associated with IgE responses to -Gal and red meat allergy.

Neurite outgrowth is mediated by dynamic changes of the cytoskeleton and is largely controlled by Rho GTPases and their regulators. Here, we show that the polarity protein Scribble controls PC12 cell neurite outgrowth in response to nerve growth factor. Scribble knockdown decreases neurite numbers and increases neurite length. This effect is linked to TrkA the cognate receptor for NGF as pharmacological inhibition of phosphorylated TrkA (pTrkA) reduces Scribble expression. Moreover, Scribble forms a complex with the MAPK components ERK1/2 in a growth factor dependent manner. In RNAi experiments where Scribble expression is efficiently depleted sustained ERK1/2 phosphorylation is reduced. Conversely, siRNA with intermediate Scribble silencing efficiency fails to match this effect indicating that ERK1/2 activation depends on basic Scribble protein levels. Finally, Scribble translocates to the plasma membrane in response to growth factor where it complexes with HRas and Rac1 suggesting that the phenotype activated by loss of Scribble may be a result of altered GTPase activity. Together, these results demonstrate a novel role for Scribble in neurite outgrowth of PC12 cells.

The mammalian tick-borne flavivirus group (MTBFG) contains viruses associated with important human and animal diseases such as encephalitis and hemorrhagic fever. In contrast to mosquito-borne flaviviruses where recombination events are frequent, the evolutionary dynamic within the MTBFG was believed to be essentially clonal. This assumption was challenged with the recent report of several homologous recombinations within the Tick-borne encephalitis virus (TBEV). We performed a thorough analysis of publicly available genomes in this group and found no compelling evidence for the previously identified recombinations. However, our results show for the first time that demonstrable recombination (i.e., with large statistical support and strong phylogenetic evidences) has occurred in the MTBFG, more specifically within the Louping ill virus lineage. Putative parents, recombinant strains and breakpoints were further tested for statistical significance using phylogenetic methods. We investigated the time of divergence between the recombinant and parental strains in a Bayesian framework. The recombination was estimated to have occurred during a window of 282 to 76 years before the present. By unravelling the temporal setting of the event, we adduce hypotheses about the ecological conditions that could account for the observed recombination.

The flavivirus genus includes important human pathogens like Tick-borne encephalitis virus (TBEV), Dengue virus (DV) and West-Nile virus (WNV), that can cause severe disease e.g. encephalitis or hemorrhagic fever. The NS5 protein is a multifunctional RNA dependent RNA polymerase indispensable for the flavivirus replication. We have previously shown that TBEVNS5 contains a unique internal PDZ binding motif (YS²²³) for specific targeting of the PDZ protein Scribble. This interaction has impact on both viral down regulation of host cellular defense systems and neurite outgrowth. Putative C-terminal PDZ binding motifs present in TBEVNS5 (-SII⁹⁰³) and WNVNS5 (-TVL⁹⁰⁵) have also previously been highlighted.

To determine whether the PDZ binding motifs of TBEVNS5 has an effect on virus replication we constructed a DNA based sub-genomic TBEV replicon expressing firefly luciferase. The motifs within NS5 were mutated individually and in concert and the replicons were assayed in cell culture. Our results show that the replication rate was impaired in all mutants, which indicates that PDZ dependent host interactions influence flavivirus replication.We also find that the C-terminal PDZ binding motif present in TBEVNS5 and WNVNS5 are targeting various human PDZ domain proteins. TBEVNS5 has high affinity to Zonulaoccludens-2 (ZO-2),GIAP C-terminus interacting protein (GIPC), Calcium/calmodulin-dependent serine protein kinase (CASK) and Interleukin 16 (IL-16).A different pattern was observed for WNVNS5 as it associated with IL-16, and several other putative interaction partners.