Two PDZ binding motifs within NS5 have roles in Tick-borne encephalitis virus replication

AbstractThe flavivirus genus includes important human neurotropic pathogens like Tick-borne encephalitis virus (TBEV) and West-Nile virus (WNV). Flavivirus replication occurs at replication complexes, where the NS5 protein provides both RNA cap methyltransferase and RNA-dependent RNA polymerase activities. TBEVNS5 contains two PDZ binding motifs (PBMs) important for specific targeting of human PDZ proteins including Scribble, an association important for viral down regulation of cellular defense systems and neurite outgrowth.To determine whether the PBMs of TBEVNS5 affects virus replication we constructed a DNA based sub-genomic TBEV replicon expressing firefly luciferase. The PBMs 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 TBEV replication. We also find that the C-terminal PBMs present in TBEVNS5 and WNVNS5 are targeting various human PDZ domain proteins. TBEVNS5 has affinity to Zonula occludens-2 (ZO-2), GIAP C-terminus interacting protein (GIPC), calcium/calmodulin-dependent serine protein kinase (CASK), glutamate receptor interacting protein 2, (GRIP2) and Interleukin 16 (IL-16). A different pattern was observed for WNVNS5 as it associate with a broader repertoire of putative host PDZ proteins

First Dating of a Recombination Event in Mammalian Tick-Borne Flaviviruses

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

Molecular characterization of the Tick-borne encephalitis virus : Environments and replication

The flavivirus genus is of major concern for world morbidity and mortality and includes viruses causing both encephalitic as well as hemorrhagic diseases. The incidence of Tick-borne encephalitis is increasing in many European countries and several reports have emphasized the expansion of the main vector, Ixodes ricinus. The pattern of vector distribution is also changing in Sweden, which makes it important to set up solid and successful strategies for detection and genetic characterization of novel Swedish TBEV strains. In this study we have generated strategies for detection of broad types of tick-borne flaviviruses in pools of I. ricinus sampled in Sweden. The positive collection on the island of Torö was used to generate a sequence of a complete TBEV genome straight from the arthropod reservoir. This cloned virus was used to construct a self-replicating DNA based sub-genomic TBEV replicon capable of expressing reporter genes. The replicon was used to study the effect of TBEV on neurite outgrowth, which revealed that the MTase domain of NS5 block the formation of the Scribble/Rac1/βPIX protein complex, impairing neurite outgrowth in neuronal growth factor induced PC12 cells. We also demonstrate that TBEV replication is affected by two PDZ binding motifs within NS5 and reveal putative PDZ binding proteins. These interactions might affect cellular pathways and might have a role in flavivirus replication. We also characterize the variable 3´ non-coding region (V3’-NCR) by in silico studies on TBEV. Analysis brings new evidence that V3’-NCR region carries an enhancer element important for different replication/translation dynamics during the viral lifecycle in mammalian and tick cells. We also propose a temperature-sensitive trans-acting riboswitch mechanism; altering the secondary RNA structures of a closed form at lower temperatures and a form open for translation at higher temperatures. This mechanism may explain the low TBEV level observed in sampled ticks.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p

Data from: First dating of a recombination event in mammalian tick-borne flaviviruses

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 period of 282 to 76 years before the present. By unravelling the temporal setting of the event, we adduce hypothese

Beast_inference1_E

Beast xml file used in Beast inference 1 for the E-gene

Figure3_a_ML

Tree file corresponding to result of the Maximum Likelihood inference reported in Figure 3a

Development of Live attenuated Langat virus infectious clone as potential new TBEV vaccine candidate : Study of cellular and humoral immune response in mice

Tick-borne encephalitis (TBE) is one of the most important tick-transmitted diseases in Europe and Asia. The incidence of TBE cases showed a remarkable elevation in recent years probably due to the geographic expansion of TBEV and its vectors, which is concerning in the absence of a specific antiviral treatment. Vaccination remains the best protective measure against TBE. However, currently available vaccines have a burdensome immunization schedule, and poor immunogenicity in the elderly, which may contribute to observed vaccine failures, i.e., TBE occurrence in vaccinated people. One aim within the Developvaccines@oru project is to develop a novel TBE vaccine that could provide improved immunogenicity using fewer doses. Our strategy is to induce an immune response at possible sites of virus infection by a modified live attenuated vaccine based on Langat virus (LGTV). Infectious clones of Langat virus (LGTV IC) based on the strain available in our laboratory are created followed by the generation of modified LGTV IC as potential attenuated virus. Then, we compare them with the ¨original¨ LGTV strain using cell based and animal models. In our laboratory, we f successfully created LGTV IC. In order to establish a baseline for animal experiments with our vaccine candidates, we planned a pilot study using the “original” LGTV and LGTV IC strains. First, we conducted a pre-pilot experiment to optimize the study design and evaluation methods. Our preliminary data shows that intramuscular administration of both strains was well tolerated in mice.  In contrast to the original LGTV, LGTV IC was found to cause a transient, but significant reduction in body weight. ELISA results showed that mice antibodies after LGTV IC infection cross reacted with TBEV antigens. The T lymphocytes, isolated from these mice spleens, showed Interferon gamma secretion when stimulated with both LGTV and TBEV peptides. However, this cellular response revealed higher in original LGTV infected mice. Moreover, we confirm that LGTV IC show lower viraemia peak than original LGTV, both occurring at 2 days post infection. Besides, we have successfully rescued modified LGTV infectious clones with individual or combined mutations in genomic regions coding for NS3, NS5 and/or 3’non-coding region of LGTV genome. Further in-vitro and in-vivo investigation of the modified and potentially attenuated LGTV clone, seems interesting in the development approach of new TBEV vaccine candidate.

Roles of the Endogenous Lunapark Protein during Flavivirus Replication

The endoplasmic reticulum (ER) of eukaryotic cells is a dynamic organelle, which undergoes continuous remodeling. At the three-way tubular junctions of the ER, the lunapark (LNP) protein acts as a membrane remodeling factor to stabilize these highly curved membrane junctions. In addition, during flavivirus infection, the ER membrane is invaginated to form vesicles (Ve) for virus replication. Thus, LNP may have roles in the generation or maintenance of the Ve during flavivirus infection. In this study, our aim was to characterize the functions of LNP during flavivirus infection and investigate the underlying mechanisms of these functions. To specifically study virus replication, we generated cell lines expressing replicons of West Nile virus (Kunjin strain) or Langat virus. By using these replicon platforms and electron microscopy, we showed that depletion of LNP resulted in reduced virus replication, which is due to its role in the generation of the Ve. By using biochemical assays and high-resolution microscopy, we found that LNP is recruited to the Ve and the protein interacts with the nonstructural protein (NS) 4B. Therefore, these data shed new light on the interactions between flavivirus and host factors during viral replication.This article belongs to the Special Issue Host Cell Organelles in Viral Infections: Friends and Foes. Academic Editors: Daniela Ribeiro and Markus Islinger.</p

Beast_inference1_C

Beast xml file used in Beast inference 1 for the C-gene

Development of a potential live-attenuated Langat virus as candidate for novel tick-borne encephalitis vaccine

Background. Tick-borne encephalitis (TBE) is one of the most important tick-transmitted diseases in Europe and Asia. Most infections with the TBE virus (TBEV) are asymptomatic or cause mild flu-like symptoms, but they may induce severe neurological disorders with permanent sequelae. The incidence of TBE cases showed a remarkable elevation in recent years probably due to the geographic expansion of TBEV and its vectors, which is concerning in the absence of a specific antiviral treatment. Vaccination remains the best protective measure against TBE. However, currently available vaccines have a burdensome immunization schedule, and poor immunogenicity in the elderly, which may contribute to observed vaccine failures, i.e., TBE occurrence in vaccinated people. One aim within the Developvaccines@oru project is to develop a novel TBE vaccine that could provide improved immunogenicity using fewer doses. Our strategy is to induce an immune response at possible sites of virus infection by a modified live attenuated vaccine based on Langat virus.  Methods. Infectious clones of Langat virus (LGTV) based on the strain available in our laboratory are created followed by the generation of modified LGTV infectious clone as potential attenuated virus. Then, we compare them with the ¨original¨ LGTV strain using cell based and animal models. Preliminary results. We successfully created LGTV infectious clones. In order to establish a baseline for animal experiments with our vaccine candidates, we planned a pilot study using the “original” Langat virus. First, we conducted a pre-pilot experiment to optimize the study design and evaluation methods. Preliminary data on the establishment process of vaccine candidates in vitro as well as cellular and humoral immune response in mice in response to the LGTV infectious clone are presented. Conclusion. Further investigation of modified LGTV clone seems interesting in the development approach of new TBEV vaccine candidate