Breast milk transmission and involvement of mammary glands in tick-borne flavivirus infected mice.

Tick-borne flaviviruses (TBFs) are transmitted to humans through milk and tick bites. Although a case of possible mother-to-child transmission of tick-borne encephalitis virus (TBEV) through breast milk has been reported, this route has not been confirmed in experimental models. Therefore, in this study, using type I interferon receptor-deficient A129 mice infected with Langat virus (LGTV), we aimed to demonstrate the presence of infectious virus in the milk and mammary glands of infected mice. Our results showed viral RNA of LGTV in the pup's stomach milk clots (SMCs) and blood, indicating that the virus can be transmitted from dam to pup through breast milk. In addition, we observed that LGTV infection causes tissue lesions in the mammary gland, and viral particles were present in mammary gland epithelial cells. Furthermore, we found that milk from infected mice could infect adult mice via the intragastric route, which has a milder infection process, longer infection time, and a lower rate of weight loss than other modes of infection. Specifically, we developed a nano-luciferase-LGTV reporter virus system to monitor the dynamics of different infection routes and observed dam-to-pup infection using in vivo bioluminescence imaging. This study provides comprehensive evidence to support breast milk transmission of TBF in mice and has helped provide useful data for studying TBF transmission routes.IMPORTANCETo date, no experimental models have confirmed mother-to-child transmission of tick-borne flavivirus (TBF) through breastfeeding. In this study, we used a mouse model to demonstrate the presence of infectious viruses in mouse breast milk and mammary gland epithelial cells. Our results showed that pups could become infected through the gastrointestinal route by suckling milk, and the infection dynamics could be monitored using a reporter virus system during breastfeeding in vivo. We believe our findings have provided substantial evidence to understand the underlying mechanism of breast milk transmission of TBF in mice, which has important implications for understanding and preventing TBF transmission in humans.

Evaluation of two artificial infection methods of live ticks as tools for studying interactions between tick-borne viruses and their tick vectors.

Up to 170 tick-borne viruses (TBVs) have been identified to date. However, there is a paucity of information regarding TBVs and their interaction with respective vectors, limiting the development of new effective and urgently needed control methods. To overcome this gap of knowledge, it is essential to reproduce transmission cycles under controlled laboratory conditions. In this study we assessed an artificial feeding system (AFS) and an immersion technique (IT) to infect Ixodes ricinus ticks with tick-borne encephalitis (TBE) and Kemerovo (KEM) virus, both known to be transmitted predominantly by ixodid ticks. Both methods permitted TBEV acquisition by ticks and we further confirmed virus trans-stadial transmission and onward transmission to a vertebrate host. However, only artificial feeding system allowed to demonstrate both acquisition by ticks and trans-stadial transmission for KEMV. Yet we did not observe transmission of KEMV to mice (IFNAR-/- or BALB/c). Artificial infection methods of ticks are important tools to study tick-virus interactions. When optimally used under laboratory settings, they provide important insights into tick-borne virus transmission cycles.

Powassan Viruses Spread Cell to Cell during Direct Isolation from Ixodes Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes.

Powassan viruses (POWVs) are neurovirulent tick-borne flaviviruses emerging in the northeastern United States, with a 2% prevalence in Long Island (LI) deer ticks (Ixodes scapularis). POWVs are transmitted within as little as 15 min of a tick bite and enter the central nervous system (CNS) to cause encephalitis (10% of cases are fatal) and long-term neuronal damage. POWV-LI9 and POWV-LI41 present in LI Ixodes ticks were isolated by directly inoculating VeroE6 cells with tick homogenates and detecting POWV-infected cells by immunoperoxidase staining. Inoculated POWV-LI9 and LI41 were exclusively present in infected cell foci, indicative of cell to cell spread, despite growth in liquid culture without an overlay. Cloning and sequencing establish POWV-LI9 as a phylogenetically distinct lineage II POWV strain circulating in LI deer ticks. Primary human brain microvascular endothelial cells (hBMECs) and pericytes form a neurovascular complex that restricts entry into the CNS. We found that POWV-LI9 and -LI41 and lineage I POWV-LB productively infect hBMECs and pericytes and that POWVs were basolaterally transmitted from hBMECs to lower-chamber pericytes without permeabilizing polarized hBMECs. Synchronous POWV-LI9 infection of hBMECs and pericytes induced proinflammatory chemokines, interferon-ß (IFN-ß) and proteins of the IFN-stimulated gene family (ISGs), with delayed IFN-ß secretion by infected pericytes. IFN inhibited POWV infection, but despite IFN secretion, a subset of POWV-infected hBMECs and pericytes remained persistently infected. These findings suggest a potential mechanism for POWVs (LI9/LI41 and LB) to infect hBMECs, spread basolaterally to pericytes, and enter the CNS. hBMEC and pericyte responses to POWV infection suggest a role for immunopathology in POWV neurovirulence and potential therapeutic targets for preventing POWV spread to neuronal compartments. IMPORTANCE We isolated POWVs from LI deer ticks (I. scapularis) directly in VeroE6 cells, and sequencing revealed POWV-LI9 as a distinct lineage II POWV strain. Remarkably, inoculation of VeroE6 cells with POWV-containing tick homogenates resulted in infected cell foci in liquid culture, consistent with cell-to-cell spread. POWV-LI9 and -LI41 and lineage I POWV-LB strains infected hBMECs and pericytes that comprise neurovascular complexes. POWVs were nonlytically transmitted basolaterally from infected hBMECs to lower-chamber pericytes, suggesting a mechanism for POWV transmission across the blood-brain barrier (BBB). POWV-LI9 elicited inflammatory responses from infected hBMEC and pericytes that may contribute to immune cell recruitment and neuropathogenesis. This study reveals a potential mechanism for POWVs to enter the CNS by infecting hBMECs and spreading basolaterally to abluminal pericytes. Our findings reveal that POWV-LI9 persists in cells that form a neurovascular complex spanning the BBB and suggest potential therapeutic targets for preventing POWV spread to neuronal compartments.

Incrimination of shrews as a reservoir for Powassan virus.

Powassan virus lineage 2 (deer tick virus) is an emergent threat to American public health, causing severe neurologic disease. Its life cycle in nature remains poorly understood. We use a host-specific retrotransposon-targeted real time PCR assay to test the hypothesis that white-footed mice, considered the main eastern U.S. reservoir of the coinfecting agent of Lyme disease, is the reservoir for deer tick virus. Of 20 virus-infected host-seeking nymphal black-legged ticks 65% fed on shrews and none on mice. The proportion of ticks feeding on shrews at a site is positively associated with prevalence of viral infection, but not the Lyme disease agent. Viral RNA is detected in the brain of one shrew. We conclude that shrews are a likely reservoir host for deer tick virus and that host bloodmeal analysis can provide direct evidence to incriminate reservoir hosts, thereby promoting our understanding of the ecology of tick-borne infections.

Vector competence of human-biting ticks Ixodes scapularis, Amblyomma americanum and Dermacentor variabilis for Powassan virus.

BACKGROUND: Powassan virus (POWV; genus Flavivirus) is the sole North American member of the tick-borne encephalitis sero-complex and an increasing public health threat in the USA. Maintained in nature by Ixodes spp. ticks, POWV has also been isolated from species of other hard tick genera, yet it is unclear if these species can serve as vectors. Dermacentor variabilis and Amblyomma americanum share geographic and ecologic overlap with Ixodes spp. ticks and POWV transmission foci, raising the possibility that POWV could become established in these tick species and leading to range expansion and increased human risk. Therefore, we assessed the competency of Ixodes scapularis, D. variabilis and A. americanum for POWV lineage II (POWV II). METHODS: Larvae from all three species were co-infested on POWV-infected Balb/c mice. The engorged larvae were allowed to molt to nymphs and screened for the presence of POWV II RNA by reverse transcription-qPCR. Eight infected nymphs from each species were allowed to individually feed on a naïve mouse. Mice were screened for the presence of POWV II RNA to determine infection status. RESULTS: The results demonstrated that larvae from all three tick species were able to efficiently acquire POWV II via feeding on viremic mice, maintain infection through molting and successively transmit POWV to naïve mice at the nymphal stage at comparable rates across all three species. CONCLUSIONS: Our findings reveal that non-Ixodes tick species can serve as competent vectors for POWV and highlight the potential role of these species in the ecology and epidemiology of POWV. Future studies examining the possible implications of these findings on POWV epidemiology and the adaptability of POWV in these new vectors are warranted.

Alkhumra hemorrhagic fever virus infection.

OBJECTIVES: This review article summarizes what has been published on Alkhumra hemorrhagic fever virus (AHFV), a novel flavivirus that was discovered in Saudi Arabia in 1995. METHODS: PubMed was used to search for studies published from January 1995 to June 2019 using the key words Alkhumra virus, Alkhurma virus, novel flavivirus, and tick-borne encephalitis virus. Additionally, records of the Saudi Ministry of Health were reviewed. RESULTS: Thirty-two articles on AHFV were identified. Acute febrile flu-like illness, hepatitis, hemorrhagic manifestations, and, less commonly, encephalitis are the main clinical features. The virus seems to be transmitted from livestock animals to humans by direct contact with these animals or their raw meat, or perhaps by tick or mosquito bites. The ability of ticks and mosquitoes to serve as vectors for AHFV needs to be confirmed by biological studies. The exact role of animals such as sheep, goats, camels, and other mammals in the transmission and maintenance of the virus remains to be elucidated. Preventive measures require an interdisciplinary approach involving the human and veterinary health sectors, the municipality, the ministry of agriculture, the vector control sector, and academic and research institutes. CONCLUSIONS: AHFV has been well characterized; nevertheless, some aspects remain to be elucidated.

Alimentary Infections by Tick-Borne Encephalitis Virus.

Tick-borne encephalitis virus (TBEV) causes serious the neurological disease, tick-borne encephalitis (TBE). TBEV can be transmitted to humans by ticks as well as by the alimentary route, which is mediated through the consumption of raw milk products from infected ruminants such as sheep, goats, and cows. The alimentary route of TBEV was recognized in the early 1950s and many important experimental studies were performed shortly thereafter. Nowadays, alimentary TBEV infections are recognized as a relevant factor contributing to the overall increase in TBE incidences in Europe. This review aims to summarize the history and current extent of alimentary TBEV infections across Europe, to analyze experimental data on virus secretion in milk, and to review possible alimentary infection preventive measures.

Migratory birds as disseminators of ticks and the tick-borne pathogens Borrelia bacteria and tick-borne encephalitis (TBE) virus: a seasonal study at Ottenby Bird Observatory in South-eastern Sweden.

BACKGROUND: Birds can act as reservoirs of tick-borne pathogens and can also disperse pathogen-containing ticks to both nearby and remote localities. The aims of this study were to estimate tick infestation patterns on migratory birds and the prevalence of different Borrelia species and tick-borne encephalitis virus (TBEV) in ticks removed from birds in south-eastern Sweden. METHODS: Ticks were collected from resident and migratory birds captured at the Ottenby Bird Observatory, Öland, Sweden, from March to November 2009. Ticks were molecularly identified to species, and morphologically to developmental stage, and the presence of Borrelia bacteria and TBEV was determined by quantitative real-time PCR. RESULTS: A total of 1339 ticks in the genera Haemaphysalis, Hyalomma, and Ixodes was recorded of which I. ricinus was the most abundant species. Important tick hosts were the European robin (Erithacus rubecula), Blackbird (Turdus merula), Tree pipit (Anthus trivialis), Eurasian wren (Troglodytes troglodytes), Common redstart (Phoenicurus phoenicurus), Willow warbler (Phylloscopus trochilus), and Common whitethroat (Sylvia communis). Borrelia bacteria were detected in 25% (285/1,124) of the detached ticks available for analysis. Seven Borrelia species (B. afzelii, B. burgdorferi (s.s.), B. garinii, B. lusitaniae, B. turdi, B. valaisiana, and B. miyamotoi) were identified. B. turdi was recorded for the first time in ticks in Sweden. The number of Borrelia cells per tick ranged from 2.0 × 100 to 7.0 × 105. B. miyamotoi-containing ticks contained a significantly higher median number of Borrelia cells than B. burgdorferi (s.l.)-containing ticks. B. garinii and B. miyamotoi were the most prevalent Borrelia species in tick larvae. Larvae of I. ricinus with B. garinii were removed from seven bird species, particularly S. communis and A. trivialis, which may suggest that the larvae had contracted the Borrelia bacteria from or via these birds. Also, a high percentage of tick larvae containing B. miyamotoi was removed from E. rubecula. All ticks were negative for TBEV. CONCLUSIONS: The results corroborate the view that the contributions of birds to human disease are substantial, particularly as blood hosts for ticks and for their short-, medium-, and long-distance dispersal. Moreover, several ground-foraging bird species appear to be important for the maintenance and dispersal of Borrelia species. The absence of TBEV in the ticks conforms to other similar studies.

Vectors, molecular epidemiology and phylogeny of TBEV in Kazakhstan and central Asia.

BACKGROUND: In the South of Kazakhstan, Almaty Oblast' (region) is endemic for tick-borne encephalitis, with 0.16-0.32 cases/100,000 population between 2016-2018. The purpose of this study was to determine the prevalence and circulating subtypes of tick-borne encephalitis virus (TBEV) in Almaty Oblast' and Kyzylorda Oblast'. METHODS: In 2015 we investigated 2341 ticks from 7 sampling sites for the presence of TBEV. Ticks were pooled in 501 pools and isolated RNA was tested for the presence of TBEV by RT-qPCR. For the positive samples, the E gene was amplified, sequenced and a phylogenetic analysis was carried out. RESULTS: A total of 48 pools were TBEV-positive by the RT-qPCR. TBEV-positive ticks were only detected in three districts of Almaty Oblast' and not in Kyzylorda Oblast'. The positive TBEV pools were found within Ixodes persulcatus, Haemaphysalis punctata and Dermacentor marginatus. These tick species prevailed only in Almaty Oblast' whereas in Kyzylorda Oblast' Hyalomma asiaticum and D. marginatus are endemic. The minimum infection rates (MIR) in the sampling sites were 4.4% in Talgar, 2.8% in Tekeli and 1.1% in Yenbekshikazakh, respectively. The phylogenetic analysis of the generated sequences indicates that TBEV strains found in Almaty Oblast' clusters in the Siberian subtype within two different clades. CONCLUSIONS: We provided new data about the TBEV MIR in ticks in Almaty Oblast' and showed that TBEV clusters in the Siberian Subtype in two different clusters at the nucleotide level. These results indicate that there are different influences on the circulating TBEV strains in south-eastern Kazakhstan. These influences might be caused by different routes of the virus spread in ticks which might bring different genetic TBEV lineages to Kazakhstan. The new data about the virus distribution and vectors provided here will contribute to an improvement of monitoring of tick-borne infections and timely anti-epidemic measures in Kazakhstan.

Acute tick-borne encephalitis during pregnancy - An alarming combination.

We report on a pregnant patient who contracted tick-borne encephalitis (TBE) during her second trimester in an endemic region in Southern Germany. The patient presented with typical symptoms including fever and headache, and TBE infection was confirmed by positive blood and cerebrospinal fluid (CSF) testing. During acute infection there was no evidence of pregnancy complications, and the mother recovered well. We performed a clinical follow-up examination of both mother and child eight months after the diagnosis of TBE, which revealed no signs of sequelae. This case study presents rare evidence of TBE infection during pregnancy and may provide guidance for both physicians as well as mothers-to-be dealing with TBE.

Tick-borne encephalitis outbreak following raw goat milk consumption in a new micro-location, Croatia, June 2019.

In June 2019, the Croatian Institute of Public Health was informed of a cluster of patients with laboratory confirmed tick-borne encephalitis (TBE) from the Gorski Kotar region. Five of the six patients with TBE reported consuming raw (unpasteurized) goat milk in the two week period before symptom onset, and one reported a recent tick bite. To assess risk factors for infection, we selected six control individuals from among healthy family and community members, and conducted a case-control analysis. None of the cases or controls were vaccinated against TBE. Individuals with TBE (cases) had 25 (95 % CI 0.8-1410.2, p = 0.021) times higher odds of raw goat milk consumption compared to healthy controls. Milk samples from 12 goats from the implicated farm were tested for the TBE virus (TBEV) using RT-PCR. TBEV RNA was not detected in the milk, but serological testing of goats and other farm animals yielded evidence of exposure to the virus: Six goats from the flock had TBEV neutralizing antibodies. Our findings suggest that the vehicle for the outbreak was raw goat milk from a single farm. Following public health advice to cease consumption of raw dairy products, no further cases have been reported.

Questing abundance of adult taiga ticks Ixodes persulcatus and their Borrelia prevalence at the north-western part of their distribution.

BACKGROUND: Because ixodid ticks are vectors of zoonotic pathogens, including Borrelia, information of their abundance, seasonal variation in questing behaviour and pathogen prevalence is important for human health. As ticks are invading new areas northwards, information from these new areas are needed. Taiga tick (Ixodes persulcatus) populations have been recently found at Bothnian Bay, Finland. We assessed seasonal variation in questing abundance of ticks and their pathogen prevalence in coastal deciduous forests near the city of Oulu (latitudes 64-65°) in 2019. METHODS: We sampled ticks from May until September by cloth dragging 100 meters once a month at eight study sites. We calculated a density index (individuals/100 m2) to assess seasonal variation. Samples were screened for Borrelia burgdorferi (sensu lato) (including B. afzelii, B. garinii, B. burgdorferi (sensu stricto) and B. valaisana), Borrelia miyamotoi, Anaplasma phagocytophilum, Rickettsia spp., Neoehrlichia mikurensis, Francisella tularensis and Bartonella spp., Babesia spp. and for the tick-borne encephalitis virus. RESULTS: All except one nymph were identified as I. persulcatus. The number of questing adults showed a strong peak in May (median: 6.5 adults/100 m2), which is among the highest values reported in northern Europe, and potentially indicates a large population size. After May, the number of questing adults declined steadily with few adults still sampled in August. Nymphs were present from May until September. We found a striking prevalence of Borrelia spp. in adults (62%) and nymphs (40%), with B. garinii (51%) and B. afzelii (63%) being the most common species. In addition, we found that 26% of infected adults were coinfected with at least two Borrelia genospecies, mainly B. garinii and B. afzelii, which are associated with different host species. CONCLUSIONS: The coastal forest environments at Bothnian Bay seem to provide favourable environments for I. persulcatus and the spread of Borrelia. High tick abundance, a low diversity of the host community and similar host use among larvae and nymphs likely explain the high Borrelia prevalence and coinfection rate. Research on the infestation of the hosts that quantifies the temporal dynamics of immature life stages would reveal important aspects of pathogen circulation in these tick populations.

Tick-borne encephalitis virus (TBEV) infection in pregnancy: Absence of virus transmission to the fetuses despite severe maternal disease - A case study.

Tick-borne encephalitis (TBE) is an emerging infectious disease in large parts of Europe and Asia. Whereas other members of the Flaviviridae family can harm fetal development, there are only very few reports on TBE virus (TBEV) infections during pregnancy. Thus, the implications for fetal health remain largely unknown. In this study, we present detailed pre- and postnatal health assessment of three children in the context of severe maternal TBEV infection during pregnancy. Following acute TBEV infection of the mothers, intrauterine growth and development of all children were assessed by repetitive prenatal ultrasound. Postnatal examinations included clinical and virological analyses over a follow-up period of 18 months. Prenatally, no signs of intrauterine growth restrictions were observed. All neonates were delivered at term. Umbilical cord blood of the newborns tested negative for TBEV RNA. Virus-specific IgG antibodies were positive at birth but negative at 9 and 11 months of age. Importantly, IgM antibodies remained negative throughout the period of observation. Taken together, these clinical and virological data strongly suggest that fetal TBEV infection did not occur, despite severe manifestations in the mothers.

Large-scale countrywide screening for tick-borne pathogens in field-collected ticks in Latvia during 2017-2019.

BACKGROUND: Tick-borne diseases are of substantial concern worldwide in both humans and animals. Several hard tick species are of medical and veterinary interest in Europe, and changes in the range of tick species can affect the spread of zoonotic pathogens. The aim of the present study was to map the current prevalence and distribution pattern of ticks and related tick-borne pathogens in Latvia, a Baltic state in northern Europe. METHODS: Nearly 4600 Ixodes ricinus, I. persulcatus and Dermacentor reticulatus tick samples were collected in all regions of Latvia during 2017-2019 and were screened by molecular methods to reveal the prevalence and distribution pattern of a wide spectrum of tick-borne pathogens. RESULTS: New localities of D. reticulatus occurrence were found in western and central Latvia, including the Riga region, indicating that the northern border of D. reticulatus in Europe has moved farther to the north. Among the analyzed ticks, 33.42% carried at least one tick-borne pathogen, and 5.55% of tick samples were positive for two or three pathogens. A higher overall prevalence of tick-borne pathogens was observed in I. ricinus (34.92%) and I. persulcatus (31.65%) than in D. reticulatus (24.2%). The molecular analysis revealed the presence of tick-borne encephalitis virus, Babesia spp., Borrelia spp., Anaplasma phagocytophilum and Rickettsia spp. Overall, 15 and 7 tick-borne pathogen species were detected in Ixodes spp. and D. reticulatus ticks, respectively. This is the first report of Borrelia miyamotoi in Latvian field-collected ticks. CONCLUSIONS: This large-scale countrywide study provides a snapshot of the current distribution patterns of Ixodes and Dermacentor ticks in Latvia and gives us a reliable overview of tick-borne pathogens in Latvian field-collected ticks.

Tick-borne encephalitis (TBE) cases are not random: explaining trend, low- and high-frequency oscillations based on the Austrian TBE time series.

BACKGROUND: Why human tick-borne encephalitis (TBE) cases differ from year to year, in some years more 100%, has not been clarified, yet. The cause of the increasing or decreasing trends is also controversial. Austria is the only country in Europe where a 40-year TBE time series and an official vaccine coverage time series are available to investigate these open questions. METHODS: A series of generalized linear models (GLMs) has been developed to identify demographic and environmental factors associated with the trend and the oscillations of the TBE time series. Both the observed and the predicted TBE time series were subjected to spectral analysis. The resulting power spectra indicate which predictors are responsible for the trend, the high-frequency and the low-frequency oscillations, and with which explained variance they contribute to the TBE oscillations. RESULTS: The increasing trend can be associated with the demography of the increasing human population. The responsible GLM explains 12% of the variance of the TBE time series. The low-frequency oscillations (10 years) are associated with the decadal changes of the large-scale climate in Central Europe. These are well described by the so-called Scandinavian index. This 10-year oscillation cycle is reinforced by the socio-economic predictor net migration. Considering the net migration and the Scandinavian index increases the explained variance of the GLM to 44%. The high-frequency oscillations (2-3 years) are associated with fluctuations of the natural TBE transmission cycle between small mammals and ticks, which are driven by beech fructification. Considering also fructification 2 years prior explains 64% of the variance of the TBE time series. Additionally, annual sunshine duration as predictor for the human outdoor activity increases the explained variance to 70%. CONCLUSIONS: The GLMs presented here provide the basis for annual TBE forecasts, which were mainly determined by beech fructification. A total of 3 of the 5 years with full fructification, resulting in high TBE case numbers 2 years later, occurred after 2010. The effects of climate change are therefore not visible through a direct correlation of the TBE cases with rising temperatures, but indirectly via the increased frequency of mast seeding.

Evaluation of Viral RNA Recovery Methods in Vectors by Metagenomic Sequencing.

Identification and characterization of viral genomes in vectors including ticks and mosquitoes positive for pathogens of great public health concern using metagenomic next generation sequencing (mNGS) has challenges. One such challenge is the ability to efficiently recover viral RNA which is typically dependent on sample processing. We evaluated the quantitative effect of six different extraction methods in recovering viral RNA in vectors using negative tick homogenates spiked with serial dilutions of tick-borne encephalitis virus (TBEV) and surrogate Langat virus (LGTV). Evaluation was performed using qPCR and mNGS. Sensitivity and proof of concept of optimal method was tested using naturally positive TBEV tick homogenates and positive dengue, chikungunya, and Zika virus mosquito homogenates. The amount of observed viral genome copies, percentage of mapped reads, and genome coverage varied among different extractions methods. The developed Method 5 gave a 120.8-, 46-, 2.5-, 22.4-, and 9.9-fold increase in the number of viral reads mapping to the expected pathogen in comparison to Method 1, 2, 3, 4, and 6, respectively. Our developed Method 5 termed ROVIV (Recovery of Viruses in Vectors) greatly improved viral RNA recovery and identification in vectors using mNGS. Therefore, it may be a more sensitive method for use in arbovirus surveillance.

Dermacentor reticulatus is a vector of tick-borne encephalitis virus.

Tick-borne encephalitis virus (TBEV; family Flaviviridae) is the most medically important tick-borne virus in Europe and Asia. Ixodes ricinus and I. persulcatus ticks are considered to be the main vector ticks of TBEV in nature due to their specific ecological associations with the vertebrate hosts. Nevertheless, recent TBEV prevalence studies in ticks suggest that Dermacentor reticulatus ticks might play a relevant role in the maintenance of TBEV in nature. The goal of this study was to evaluate the vector competency of D. reticulatus for TBEV through experimental tick infections and comparative in vivo transmission studies involving D. reticulatus and I. ricinus ticks. We observed that after a transcoxal micro-capillary inoculation, adult female D. reticulatus ticks efficiently replicated TBEV during the observed period of 21 days. The mean virus load reached up to 2.5 × 105 gene copies and 6.4 × 104 plaque forming units per tick. The infected D. reticulatus ticks were able to transmit the virus to mice. The course of infection in mice was comparable to the infection after a tick bite by I. ricinus while the virus spread and clearance was slightly faster. Moreover, D. reticulatus ticks were capable of tick-to-tick non-viraemic transmission of TBEV to the Haemaphysalis inermis nymphs during co-feeding on the same animal. The co-feeding transmission efficiency was overall slightly lower (up to 54 %) in comparison with I. ricinus (up to 94 %) and peaked 1 day later, at day 3. In conclusion, our study demonstrated that D. reticulatus is a biologically effective vector of TBEV. In line with the recent reports of its high TBEV prevalence in nature, our data indicate that in some endemic foci, D. reticulatus might be an underrecognized TBEV vector which contributes to the expansion of the TBEV endemic areas.

Tick-Borne Encephalitis Virus: An Emerging Ancient Zoonosis?

Tick-borne encephalitis (TBE) is one of the most important viral zoonosis transmitted by the bite of infected ticks. In this study, all tick-borne encephalitis virus (TBEV) E gene sequences available in GenBank as of June 2019 with known date of isolation (n = 551) were analyzed. Simulation studies showed that a sample bias could significantly affect earlier studies, because small TBEV datasets (n = 50) produced non-overlapping intervals for evolutionary rate estimates. An apparent lack of a temporal signal in TBEV, in general, was found, precluding molecular clock analysis of all TBEV subtypes in one dataset. Within all subtypes and most of the smaller groups in these subtypes, there was evidence of many medium- and long-distance virus transfers. These multiple random events may play a key role in the virus spreading. For some groups, virus diversity within one territory was similar to diversity over the whole geographic range. This is best exemplified by the virus diversity observed in Switzerland or Czech Republic. These two countries yielded most of the known European subtype Eu3 subgroup sequences, and the diversity of viruses found within each of these small countries is comparable to that of the whole Eu3 subgroup, which is prevalent all over Central and Eastern Europe. Most of the deep tree nodes within all three established TBEV subtypes dated less than 300 years back. This could be explained by the recent emergence of most of the known TBEV diversity. Results of bioinformatics analysis presented here, together with multiple field findings, suggest that TBEV may be regarded as an emerging disease.

The potential impact of climate change on the transmission risk of tick-borne encephalitis in Hungary.

BACKGROUND: Impact of climate change on tick-borne encephalitis (TBE) prevalence in the tick-host enzootic cycle in a given region depends on how the region-specific climate change patterns influence tick population development processes and tick-borne encephalitis virus (TBEV) transmission dynamics involving both systemic and co-feeding transmission routes. Predicting the transmission risk of TBEV in the enzootic cycle with projected climate conditions is essential for planning public health interventions including vaccination programs to mitigate the TBE incidence in the inhabitants and travelers. We have previously developed and validated a mathematical model for retroactive analysis of weather fluctuation on TBE prevalence in Hungary, and we aim to show in this research that this model provides an effective tool for projecting TBEV transmission risk in the enzootic cycle. METHODS: Using the established model of TBEV transmission and the climate predictions of the Vas county in western Hungary in 2021-2050 and 2071-2100, we quantify the risk of TBEV transmission using a series of summative indices - the basic reproduction number, the duration of infestation, the stage-specific tick densities, and the accumulated (tick) infections due to co-feeding transmission. We also measure the significance of co-feeding transmission by observing the cumulative number of new transmissions through the non-systemic transmission route. RESULTS: The transmission potential and the risk in the study site are expected to increase along with the increase of the temperature in 2021-2050 and 2071-2100. This increase will be facilitated by the expected extension of the tick questing season and the increase of the numbers of susceptible ticks (larval and nymphal) and the number of infected nymphal ticks co-feeding on the same hosts, leading to compounded increase of infections through the non-systemic transmission. CONCLUSIONS: The developed mathematical model provides an effective tool for predicting TBE prevalence in the tick-host enzootic cycle, by integrating climate projection with emerging knowledge about the region-specific tick ecological and pathogen enzootic processes (through model parametrization fitting to historical data). Model projects increasing co-feeding transmission and prevalence of TBEV in a recognized TBE endemic region, so human risk of TBEV infection is likely increasing unless public health interventions are enhanced.

Tick-Borne Encephalitis Virus, United Kingdom.

During February 2018-January 2019, we conducted large-scale surveillance for the presence and prevalence of tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) in sentinel animals and ticks in the United Kingdom. Serum was collected from 1,309 deer culled across England and Scotland. Overall, 4% of samples were ELISA-positive for the TBEV serocomplex. A focus in the Thetford Forest area had the highest proportion (47.7%) of seropositive samples. Ticks collected from culled deer within seropositive regions were tested for viral RNA; 5 of 2,041 ticks tested positive by LIV/TBEV real-time reverse transcription PCR, all from within the Thetford Forest area. From 1 tick, we identified a full-length genomic sequence of TBEV. Thus, using deer as sentinels revealed a potential TBEV focus in the United Kingdom. This detection of TBEV genomic sequence in UK ticks has important public health implications, especially for undiagnosed encephalitis.