No detection of tick-borne encephalitis virus RNA in blood, urine or saliva of hospitalised immunocompetent tick-borne encephalitis patients.

Tick-borne encephalitis (TBE) is usually diagnosed based on the presence of TBE virus (TBEV)-specific IgM and IgG antibodies in serum. However, antibodies induced by vaccination or cross-reactivity to previous flavivirus infections may result in false positive TBEV serology. Detection of TBEV RNA may be an alternative diagnostic approach to detect viral presence and circumvent the diagnostic difficulties present when using serology. Viral RNA in blood is commonly detectable only in the first viremic phase usually lasting up to two weeks, and not in the second neurologic phase, when the patients contact the health care system and undergo diagnostic work-up. TBEV RNA has previously been detected in urine in a few retrospective TBE cases in the neurologic phase, and furthermore RNA of other flaviviruses has been detected in patient saliva. In this study, blood, saliva and urine were collected from 31 hospitalised immunocompetent patients with pleocytosis and symptoms of aseptic meningitis and/or encephalitis, suspected to have TBE. We wanted to pursue if molecular testing of TBEV RNA in these patient materials may be useful in the diagnostics. Eleven of the 31 study patients were diagnosed with TBE based on ELISA detection of TBEV specific IgG and IgM antibodies. None of the study patients had TBEV RNA detectable in any of the collected patient material.

Serological screening for tick-borne encephalitis virus in eight Norwegian herds of semi-domesticated reindeer (Rangifer tarandus tarandus).

Tick-borne encephalitis virus (TBEV) is found in Ixodes ricinus ticks throughout the area where viable tick populations exist. In Norway, TBEV is found in I. ricinus from the south coast until Brønnøy municipality in Nordland County and the range of the vector is expanding due to changes in climate, vegetation, host animals and environmental conditions. TBEV might thus have the potential to establish in new areas when I. ricinus expand its geographical distribution. At present, there is little knowledge on the status of the virus in high-altitude areas of inland regions in Norway. It has previously been indicated that reindeer may be an important sentinel species and indicator of the spread of ticks and TBEV in high-altitude regions. In this study, 408 semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus) from eight herds, from Tana in Troms and Finnmark County in northern Norway to Filefjell in Innlandet and Viken Counties in southern Norway, were screened for TBEV antibodies using a commercial enzyme-linked immunosorbent assay (ELISA). We found 16 TBEV reactive reindeer samples by ELISA; however, these results could not be confirmed by the serum neutralization test (SNT). This could indicate that a flavivirusand not necessarily TBEV, may be circulating among Norwegian semi-domesticated reindeer. The results also indicate that TBEV was not enzootic in Norwegian semi-domesticated reindeer in 2013-2015. This knowledge is important as an information base for future TBEV and flavivirus surveillance in Norway.

Prevalence of tick-borne encephalitis virus in questing Ixodes ricinus nymphs in southern Scandinavia and the possible influence of meteorological factors.

Ixodes ricinus ticks are Scandinavia's main vector for tick-borne encephalitis virus (TBEV), which infects many people annually. The aims of the present study were (i) to obtain information on the TBEV prevalence in host-seeking I. ricinus collected within the Øresund-Kattegat-Skagerrak (ØKS) region, which lies in southern Norway, southern Sweden and Denmark; (ii) to analyse whether there are potential spatial patterns in the TBEV prevalence; and (iii) to understand the relationship between TBEV prevalence and meteorological factors in southern Scandinavia. Tick nymphs were collected in 2016, in southern Scandinavia, and screened for TBEV, using pools of 10 nymphs, with RT real-time PCR, and positive samples were confirmed with pyrosequencing. Spatial autocorrelation and cluster analysis was performed with Global Moran's I and SatScan to test for spatial patterns and potential local clusters of the TBEV pool prevalence at each of the 50 sites. A climatic analysis was made to correlate parameters such as minimum, mean and maximum temperature, relative humidity and saturation deficit with TBEV pool prevalence. The climatic data were acquired from the nearest meteorological stations for 2015 and 2016. This study confirms the presence of TBEV in 12 out of 30 locations in Denmark, where six were from Jutland, three from Zealand and two from Bornholm and Falster counties. In total, five out of nine sites were positive from southern Sweden. TBEV prevalence of 0.7%, 0.5% and 0.5%, in nymphs, was found at three sites along the Oslofjord (two sites) and northern Skåne region (one site), indicating a potential concern for public health. We report an overall estimated TBEV prevalence of 0.1% in questing I. ricinus nymphs in southern Scandinavia with a region-specific prevalence of 0.1% in Denmark, 0.2% in southern Sweden and 0.1% in southeastern Norway. No evidence of a spatial pattern or local clusters was found in the study region. We found a strong correlation between TBEV prevalence in ticks and relative humidity in Sweden and Norway, which might suggest that humidity has a role in maintaining TBEV prevalence in ticks. TBEV is an emerging tick-borne pathogen in southern Scandinavia, and we recommend further studies to understand the TBEV transmission potential with changing climate in Scandinavia.

Transient Expression of Flavivirus Structural Proteins in Nicotiana benthamiana.

Flaviviruses are a threat to public health and can cause major disease outbreaks. Tick-borne encephalitis (TBE) is caused by a flavivirus, and it is one of the most important causes of viral encephalitis in Europe and is on the rise in Sweden. As there is no antiviral treatment available, vaccination remains the best protective measure against TBE. Currently available TBE vaccines are based on formalin-inactivated virus produced in cell culture. These vaccines must be delivered by intramuscular injection, have a burdensome immunization schedule, and may exhibit vaccine failure in certain populations. This project aimed to develop an edible TBE vaccine to trigger a stronger immune response through oral delivery of viral antigens to mucosal surfaces. We demonstrated successful expression and post-translational processing of flavivirus structural proteins which then self-assembled to form virus-like particles in Nicotiana benthamiana. We performed oral toxicity tests in mice using various plant species as potential bioreactors and evaluated the immunogenicity of the resulting edible vaccine candidate. Mice immunized with the edible vaccine candidate did not survive challenge with TBE virus. Interestingly, immunization of female mice with a commercial TBE vaccine can protect their offspring against TBE virus infection.

High-throughput sequencing of two European strains of tick-borne encephalitis virus (TBEV), Hochosterwitz and 1993/783.

Tick-borne encephalitis virus (TBEV) is a medically important arbovirus, widespread in Europe and Asia. The virus is primarily transmitted to humans and animals by bites from ticks and, in rare cases, by consumption of unpasteurized dairy products. The aim of this study was to sequence and characterize two TBEV strains with amplicon sequencing by designing overlapping primers. The amplicon sequencing, via Illumina MiSeq, covering nearly the entire TBEV genome, was successful: We retrieved and characterized the complete polyprotein sequence of two TBEV strains, Hochosterwitz and 1993/783 from Austria and Sweden, respectively. In this study the previous phylogenetic analysis of both strains was confirmed to be of the European subtypes of TBEV (TBEV-Eu) by whole genome sequencing. The Hochosterwitz strain clustered with the two strains KrM 93 and KrM 213 from South Korea, and the 1993/783 strain clustered together with the NL/UH strain from the Netherlands. Our study confirms the suitability and rapidness of the high-throughput sequencing method used to produce complete TBEV genomes from TBEV samples of high viral load giving high-molecular-weight cDNA with large overlapping amplicons.

Distribution of Neoehrlichia mikurensis in Ixodes ricinus ticks along the coast of Norway: The western seaboard is a low-prevalence region.

Neoehrlichia mikurensis is a tick-borne pathogen widespread among ticks and rodents in Europe and Asia. A previous study on Ixodes ricinus ticks in Norway suggested that N. mikurensis was scarce or absent on the south-west coast of Norway, but abundant elsewhere. The aim of this study was to further investigate the prevalence and distribution of N. mikurensis along the western seaboard of Norway in comparison with more eastern and northern areas. The second aim of the study was to examine seasonal variation of the bacterium in one specific location in the south-eastern part of Norway. Questing I. ricinus were collected from 13 locations along the coast of Norway, from Brønnøysund in Nordland County to Spjaerøy in Østfold County. In total, 11,113 nymphs in 1,113 pools and 718 individual adult ticks were analysed for N. mikurensis by real-time PCR. The mean prevalence of N. mikurensis in adult ticks was 7.9% while the estimated pooled prevalence in nymphs was 3.5%. The prevalence ranged from 0% to 25.5%, with the highest prevalence in the southernmost and the northernmost locations. The pathogen was absent, or present only at low prevalence (<5%), at eight locations, all located in the west, from 58.9°N to 64.9°N. The prevalence of N. mikurensis was significantly different between counties (p < .0001). No significant seasonal variation of N. mikurensis prevalence was observed in the period May to October 2015. Our results confirm earlier findings of a low prevalence of N. mikurensis in the western seaboard of Norway.

Cervids as sentinel-species for tick-borne encephalitis virus in Norway - A serological study.

Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE). TBEV is one of the most important neurological pathogens transmitted by tick bites in Europe. The objectives of this study were to investigate the seroprevalence of TBE antibodies in cervids in Norway and the possible emergence of new foci, and furthermore to evaluate if cervids can function as sentinel animals for the distribution of TBEV in the country. Serum samples from 286 moose, 148 roe deer, 140 red deer and 83 reindeer from all over Norway were collected and screened for TBE immunoglobulin G (IgG) antibodies with a modified commercial enzyme-linked immunosorbent assay (ELISA) and confirmed by TBEV serum neutralisation test (SNT). The overall seroprevalence against the TBEV complex in the cervid specimens from Norway was 4.6%. The highest number of seropositive cervids was found in south-eastern Norway, but seropositive cervids were also detected in southern- and central Norway. Antibodies against TBEV detected by SNT were present in 9.4% of the moose samples, 1.4% in red deer, 0.7% in roe deer, and nil in reindeer. The majority of the positive samples in our study originated from areas where human cases of TBE have been reported in Norway. The study is the first comprehensive screening of cervid species in Norway for antibodies to TBEV, and shows that cervids are useful sentinel animals to indicate TBEV occurrence, as supplement to studies in ticks. Furthermore, the results indicate that TBEV might be spreading northwards in Norway. This information may be of relevance for public health considerations and supports previous findings of TBEV in ticks in Norway.

Author Correction: Spatial data of Ixodes ricinus instar abundance and nymph pathogen prevalence, Scandinavia, 2016-2017.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Spatial data of Ixodes ricinus instar abundance and nymph pathogen prevalence, Scandinavia, 2016-2017.

Ticks carry pathogens that can cause disease in both animals and humans, and there is a need to monitor the distribution and abundance of ticks and the pathogens they carry to pinpoint potential high risk areas for tick-borne disease transmission. In a joint Scandinavian study, we measured Ixodes ricinus instar abundance at 159 sites in southern Scandinavia in August-September, 2016, and collected 29,440 tick nymphs at 50 of these sites. We additionally measured abundance at 30 sites in August-September, 2017. We tested the 29,440 tick nymphs in pools of 10 in a Fluidigm real-time PCR chip to screen for 17 different tick-associated pathogens, 2 pathogen groups and 3 tick species. We present data on the geolocation, habitat type and instar abundance of the surveyed sites, as well as presence/absence of each pathogen in all analysed pools from the 50 collection sites and individual prevalence for each site. These data can be used alone or in combination with other data for predictive modelling and mapping of high-risk areas.

Spatial patterns of pathogen prevalence in questing Ixodes ricinus nymphs in southern Scandinavia, 2016.

Tick-borne pathogens cause diseases in animals and humans, and tick-borne disease incidence is increasing in many parts of the world. There is a need to assess the distribution of tick-borne pathogens and identify potential risk areas. We collected 29,440 tick nymphs from 50 sites in Scandinavia from August to September, 2016. We tested ticks in a real-time PCR chip, screening for 19 vector-associated pathogens. We analysed spatial patterns, mapped the prevalence of each pathogen and used machine learning algorithms and environmental variables to develop predictive prevalence models. All 50 sites had a pool prevalence of at least 33% for one or more pathogens, the most prevalent being Borrelia afzelii, B. garinii, Rickettsia helvetica, Anaplasma phagocytophilum, and Neoehrlichia mikurensis. There were large differences in pathogen prevalence between sites, but we identified only limited geographical clustering. The prevalence models performed poorly, with only models for R. helvetica and N. mikurensis having moderate predictive power (normalized RMSE from 0.74-0.75, R2 from 0.43-0.48). The poor performance of the majority of our prevalence models suggest that the used environmental and climatic variables alone do not explain pathogen prevalence patterns in Scandinavia, although previously the same variables successfully predicted spatial patterns of ticks in the same area.

Geographical distribution and prevalence of tick-borne encephalitis virus in questing Ixodes ricinus ticks and phylogeographic structure of the Ixodes ricinus vector in Norway.

The tick-borne encephalitis virus (TBEV), a zoonotic flaviviral infection, is endemic in large parts of Norway and Eurasia. Humans are mainly infected with TBEV via bites from infected ticks. In Norway, the main geographical distribution of ticks is along the Norwegian coastline from southeast (~59°N) and up to the southern parts of Nordland County (~65°N). In this study, we collected ticks by flagging along the coast from Østfold County to Nordland County. By whole-genome sequencing of the mitochondrial genome of Ixodes ricinus, the phylogenetic tree suggests that there is limited phylogeographic structure both in Norway and in Europe. The overall TBEV prevalence is 0.3% for nymphs and 4.3% for adults. The highest estimated TBEV prevalence in adult ticks was detected in Rogaland and Vestfold County, while for nymphs it is highest in Vestfold, Vest-Agder and Rogaland. The present work is one of the largest studies on distribution and prevalence of TBEV in ticks in Scandinavia, showing that the virus is wider distributed in Norway than previously anticipated.

Experimental infection of lambs with tick-borne encephalitis virus and co-infection with Anaplasma phagocytophilum.

Tick-borne encephalitis virus (TBEV) is a zoonotic pathogen which may cause tick-borne encephalitis (TBE) in humans and animals. More than 10,000 cases of TBE are reported annually in Europe and Asia. However, the knowledge on TBE in animals is limited. Co-infection with Anaplasma phagocytophilum and louping ill virus (LIV), a close relative to TBEV, in sheep has been found to cause more severe disease than single LIV or A. phagocytophilum infection. The aim of this study was to investigate TBEV infection and co-infection of TBEV and A. phagocytophilum in lambs. A total of 30 lambs, aged five to six months, were used. The experiment was divided into two. In part one, pre- and post-infection of TBEV and A. phagocytophilum was investigated (group 1 to 4), while in part two, co-infection of TBEV and A. phagocytophilum was investigated (group 5 and 6). Blood samples were drawn, and rectal temperature was measured daily. Lambs inoculated with TBEV displayed no clinical symptoms, but had a short or non-detectable viremia by reverse transcription real-time PCR. All lambs inoculated with TBEV developed neutralizing TBEV antibodies. Our study is in accordance with previous studies, and indicates that TBEV rarely causes symptomatic disease in ruminants. All lambs inoculated with A. phagocytophilum developed fever and clinical symptoms of tick-borne fever, and A. phagocytophilum was present in the blood samples of all infected lambs, shown by qPCR. Significantly higher mean TBEV titer was detected in the group co-infected with TBEV and A. phagocytophilum, compared to the groups pre- or post-infected with A. phagocytophilum. These results indicate that co-infection with TBEV and A. phagocytophilum in sheep stimulates an increased TBEV antibody response.

Tick-borne encephalitis virus in cows and unpasteurized cow milk from Norway.

Tick-borne encephalitis virus (TBEV) is recognized as the most important zoonotic tick-transmitted virus in Europe. TBEV is mainly transmitted to humans through bites from TBEV-infected ticks (Ixodes ricinus and Ixodes persulcatus). However, alimentary infection after consumption of unpasteurized milk and cheese from domestic ruminants has been reported. There is little information about TBEV in ruminants in Norway. The objectives of this study were to analyse unpasteurized cow milk for TBEV RNA and to study the presence of IgG antibodies to TBEV in the same animals. A total of 112 milk and blood samples were collected from cows from five different farms spread from southern to northern Norway. The milk samples were analysed by an in-house reverse transcription (RT) real-time polymerase chain reaction and confirmed by pyrosequencing. Serum samples were screened by a commercial enzyme-linked immunosorbent assay and verified by a TBEV-specific serum neutralization test. We found TBEV RNA in unpasteurized milk collected from farms in the municipalities of Mandal, Skedsmo and Brønnøy in 5.4% of the tested animals. Specific antibodies to TBEV were only detected in Arendal, where 88.2% of the tested animals were positive. Further studies on milk containing TBEV RNA should be performed to conclude if TBEV found in unpasteurized milk in Norway is infectious, which could be of great importance in a One Health perspective.

Tick-borne encephalitis virus, Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis in Ixodes ricinus ticks collected from recreational islands in southern Norway.

The aim of this study was to determine the occurrence of tick-borne pathogens of medical importance in questing ticks collected from five recreationally used islands along the Norwegian coastline. Furthermore, since coinfection may affect the disease severity, this study aimed to determine the extent of coinfection in individual ticks or co-localization of tick-borne pathogens. In all, 4158 questing Ixodes ricinus ticks were analyzed. For detection of tick-borne encephalitis virus (TBEV), nymphs (3690) were analyzed in pools of ten. To detect Borrelia burgdorferi sensu lato, B. miyamotoi, Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis, 468 nymphs were analyzed individually. A total of five nymph pools was infected with TBEV, giving an overall prevalence of 0.14%. In the individually analyzed ticks, B. burgdorferi s. l. (15.6%), Candidatus N. mikurensis (11%), A. phagocytophilum (1.4%) and B. miyamotoi (0.9%) were detected. Coinfection was found in 3.3% of the ticks, and the only dual infection observed was with B. afzelii and Candidatus N. mikurensis. This association was significantly higher than what would occur by random chance.

Prevalence of tick-borne encephalitis virus in Ixodes ricinus ticks from three islands in north-western Norway.

Tick-borne encephalitis (TBE) is the most important viral tick-borne disease in Europe and can cause severe disease in humans. In Norway, human cases have been reported only from the southern coast. The aim of this study was to investigate the prevalence of tick-borne encephalitis virus (TBEV) in questing Ixodes ricinus ticks from the north-western part of Norway. A total of 4509 ticks were collected by flagging in May and June 2014. A subpopulation of 2220 nymphs and 162 adult ticks were analysed by real-time PCR and positive samples were confirmed by pyrosequencing. The estimated prevalence of TBEV was 3.08% among adult ticks from Sekken in Møre og Romsdal County and 0.41% among nymphs from both Hitra and Frøya in Sør-Trøndelag County. This study indicates that TBEV might be more widespread than the distribution of reported human cases suggests.