Investigation of tick-borne encephalitis virus in raw ruminant milk in Turkiye using real-time PCR.

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.

diagnosed cases ranged from July 20 to October 4, 2009. Cases peaked in early September and subsided in early October. The median age of patients with confi rmed or probable disease was 47.3 years (range 3-96 years). Infections occurred in all age groups, but most infections occurred among persons 41 to 65 years of age; 118 (60.2%) were women, and 172 were farm workers. Confi rmed and clinically diagnosed cases occurred in 18 villages, which were part of 7 towns. Most cases (182) were reported in Yiting, where the fi rst case was confi rmed, and in particular, were in persons who lived in the villages of Qingsu, 100 cases; Fantianzhu, 49 cases; Xitian, 19 cases; Shangzhai, 4 cases; and Xiateng, 4 cases.

Development of a quantitative real-time RT-PCR assay with internal control for the laboratory detection of tick borne encephalitis virus (TBEV) RNA.

Natural foci of tick-borne infections associated with tick-borne encephalitis virus (TBEV), borrelia (Borrelia burgdorferi sensu lato), including Borrelia miyamotoi, anaplasma, ehrlichia and rickettsia are widespread in the Primorsky Krai. The carriers of these pathogens are ixodid ticks. The population of Primorsky Krai meets with ticks in natural biotopes, as well as in anthropurgic foci. The aim of the study is to give a comparative assessment of the epizootic activity in the natural foci of transmissible tick-borne infections in the south of Primorsky Krai in the epidemic seasons of 2017–2020. In this periodixodid ticks (3778 samples), taken from humans in natural foci in the Primorsky Territory, were studied. The TBEV antigen was determined by enzyme-linked immunosorbent assay (ELISA), genetic markers of pathogens were detected by real-time polymerase chain reaction (RT-PCR). The incidence of tick-borne infections has been analyzed. Based on the results of monitoring the infection of ixodid ticks, we found that 26.9 % of the studied samples were infected with various pathogens of tick-borne infections. Low infection rate of TBEV in ixodid ticks and high infection with pathogens of a bacterial nature were established. TBEV antigen was detected in 30 cases (1.4 %), TBEV RNA was detected in 20 cases (0.7 %), Borrelia burgdorferis. l. DNA — in 820 cases (30.7 %), ehrlichia — in 64 (2.4 %), anaplasma — in 55 (2.1 %).Genetic marker of Rickettsia spp. was detected in 3 cases (0.9 %) out of 322 examined ixodid ticks, B. miyamotoi — in 26 cases (6.9 %) out of 373 examined samples. A positive correlation was noted between the incidence rates and the cases of detection of TBEV and Lyme borreliosis in ticks. The results obtained indicate the need for annual epidemiological monitoring of infections transmitted by ixodid ticks, to determine the real epidemic situation and the activity of the functioning natural foci transmissible tick-borne infections on the territory of Primorsky Krai.

Tick-borne encephalitis outbreaks have been reported in Europe after consumption of raw milk products from infected animals. While molecular methods are commonly used in viral foodborne outbreak investigations due to their sensitivity, specificity and rapidity, there are very few methods to detect infectious tick-borne encephalitis virus (TBEV) in milk products for routine use/analyses. To address this gap, we developed a cell culture-based method to detect infectious TBEV in artificially contaminated raw goat milk and raw goat cheese, and evaluated the sensitivity of TBEV infectivity assays. Raw goat milk samples were spiked with TBEV to achieve inoculation levels ranging from 106 to 100 TCID50/mL, and Faisselle and Tomme cheese samples were spiked so their TBEV concentrations ranged from 9.28 × 105 to 9.28 × 101 TCID50 per 2.5g. To detect infectious TBEV, Vero cells were infected by raw goat milk. For cheese samples, after homogenisation and membrane filtration, Vero cells were infected with samples adsorbed on the filter (method A) or with samples eluted from the filter (method B). After 5 days, cytopathic effects (CPEs) were observed and TBEV replication in Vero cells was confirmed by an increase in the number of genome copies/mL that were detected in cell supernatant. Infected Vero cells exhibited CPEs for both milk and cheese samples. Infectious TBEV was detected to 103 TCID50/mL in raw milk samples and to 9.28 × 101 TCID50 from Faisselle samples using both methods A and B. For Tomme samples, method A was able to detect TBEV to 9.28 × 102 TCID50/2.5g and method B to 9.28 × 103 TCID50/2.5g. The number of positive samples detected was slightly higher with method A than with method B. To conclude, this qualitative cell culture-based method can detect infectious TBEV artificially inoculated into raw milk and cheese; it should be further evaluated during foodborne outbreak investigations to detect infectious TBEV from naturally contaminated milk and cheese.

Tick-borne encephalitis (TBE) diagnosis is mainly based on serology, which however can be difficult to interpret in previously TBE-vaccinate patients and in immunosuppressed patients. The objectives of this study were to investigate if the detection of tick-borne encephalitis virus (TBEV) RNA in cerebrospinal fluid (CSF) can be used to diagnose TBE, and to assess the relation of TBEV RNA positivity to clinical characteristics and disease severity. A retrospective study was conducted in TBE patients from Region Stockholm and Uppsala, Sweden, 2015–2020. CSF-samples were analysed for TBEV RNA using RT-PCR. Data on clinical parameters, retrieved from medical records, were analysed based on TBEV RNA positivity in CSF. CSF from 36 TBE patients were analysed for the presence of TBEV RNA. TBEV RNA was detected in twenty of these patients (56%). Patients with detectable TBEV RNA in CSF had a more severe disease, with a higher rate of intensive care and assisted ventilation treatment. Overall, the mortality was 40% in patients with detectable TBEV RNA in CSF. In a subgroup of 8 patients with immunosuppressive treatment and detectable TBEV RNA in CSF, 7 (88%) had a fatal outcome. TBEV RT-PCR can be used as a complement for diagnostics in patients with underlying comorbidities involving the immune system or immunosuppressive treatment. The presence of TBEV RNA in CSF may correlate to severe infection and an increased risk of fatal outcome.

Tick-borne encephalitis (TBE) virus is considered the medically most important arthropod-borne virus in Europe. Although TBE is endemic throughout central Europe, ticks and rodents determine its maintenance in small, difficult-to-assess, natural foci. We investigated the interrelation between the population genetics of the main TBE virus (TBEV) vector tick (Ixodes ricinus), the most important reservoir host (Myodes glareolus, syn. Clethrionomys glareolus), and TBEV. Rodents and ticks were sampled on 15 sites within an exploratory study area, which has been screened regularly for TBEV occurrence in ticks for more than 10 years. On all 15 sites, ticks and bank voles were sampled, screened for TBEV presence via serology and RT-PCR, and genetically examined. Moreover, TBEV isolates derived from these analyses were sequenced. In long-term TBEV foci bank vole populations show extraordinary genetic constitutions, leading to a particular population structure, whereas ticks revealed a panmictic genetic structure overall sampling sites. Landscape genetics and habitat connectivity modeling (analysis of isolation by resistance) showed no landscape-related barriers explaining the genetic structure of the bank vole populations. The results suggest that bank voles do not simply serve as TBEV reservoirs, but their genetic composition appears to have a significant influence on establishing and maintaining long-term natural TBEV foci, whereas the genetic structure of TBEV's main vector I. ricinus does not play an important role in the sustainability of long-term TBEV foci. A thorough investigation of how and to which extent TBEV and M. glareolus genetics are associated is needed to further unravel the underlying mechanisms.

The altitudinal shift in the limit of Ixodes ricinus occurrence above the previously established altitude of 750 m above sea level has been monitored over the long-term (2002-2008) in the Krkonose Mts. (Giant Mts.), the highest in the Czech Republic, along two vertical transects in their eastern and central parts (600-1020 and 600-1270 m). Ticks were collected by flagging three times annually, and examined individually by PCR or RT-PCR for the presence of Borrelia burgdorferi sensu lato or tick-borne encephalitis virus (TBEV). A total of 5999 I. ricinus ticks were tested. TBEV RNA was detected in 26 ticks at up to 1140 m. Demonstration of TBEV in two larvae of I. ricinus indicates transovarial transmission. Similar infection rates in larvae and nymphs show vertical transmission in TBEV circulation to be very important under these mountain conditions. B. burgdorferi sensu stricto was found at up to 1040-1065 m, Borrelia garinii and Borrelia afzelii up to 1080-1140 m, and Borrelia valaisiana up to 1270 m. The total infection rates of nymphs and larvae were 7.3% and 2%, respectively. B. garinii was the most prevalent (37%), followed by B. afzelii (29%), B. burgdorferi s.s. (11%), and B. valaisiana (9%). Double to quadruple coinfections were detected in 32% of the infected ticks, most frequently B. garinii/B. afzelii. Predominance of B. garinii and B. valaisiana over B. afzelii suggests that small passerine birds moving on the ground are responsible for permanent local populations of I. ricinus in mountain localities with low numbers of small terrestrial mammals. The detection of B. burgdorferi sensu lato and TBEV in host-seeking larvae indicates an autochthonic infection. Upon analysis of the local climate we consider climate warming to be responsible for the spreading of ticks and tick-transmitted pathogens to higher altitudes.

Surveillance of tick-borne encephalitis virus in wild birds and ticks in Tomsk city and its suburbs (Western Siberia)

Prevalence of tick-borne encephalitis virus in questing Dermacentor reticulatus and Ixodes ricinus ticks in Lithuania

Tick-borne encephalitis (TBE) virus prevalence and virus genome characterization in field-collected ticks (Ixodes ricinus) from risk, non-risk and former risk areas of TBE, and in ticks removed from humans in Germany

We aimed to 1) detect tick-borne encephalitis virus (TBEV) RNA in clinical samples from patients with TBE, 2) characterise the detected RNA using Sanger sequencing, and 3) examine whether RNA detection was associated with disease severity. We studied 137 patients infected and diagnosed with TBE between 2016 and 2021 in Region Örebro County and Region Värmland. Biobanked serum (n = 129) and cerebrospinal fluid (CSF; n = 110) samples were analysed. Serum was tested for TBEV-specific antibodies, and both serum and CSF for TBEV RNA using PCR. Following nested PCR, the 5' non-coding region (5'NCR) of five samples underwent Sanger sequencing. Disease severity was assessed based on intensive care unit (ICU) admission, duration of ICU stay and need for mechanical ventilation. TBEV RNA was detected in 5 serum samples (3.9 %) and 7 CSF samples (6.4 %), representing 10 patients (7.3 %). Patients with detectable RNA were older, more frequently admitted to an ICU (p = 0.04), and more often required mechanical ventilation (p = 0.01) compared to those without detectable TBEV RNA. Sequencing of the 5'NCR in four patients revealed differences from the 5 ´NCR of the Swedish reference strain Torö-2003. The Örebro sequences were identical but differed from the Värmland sequences at two nucleotide positions. TBEV RNA was detectable in both serum and CSF of TBE patients, and its presence was associated with more frequent ICU admission and need for mechanical ventilation. Sequencing of the 5'NCR revealed genetic variation between TBEV sequences from patients in Örebro and Värmland.

Tick-borne encephalitis virus (TBEV) causes one of the most important flavivirus infections of the central nervous system, affecting humans in Europe and Asia. It is mainly transmitted by the bite of an infected tick and circulates among them and their vertebrate hosts. Until now, TBE risk analysis in Germany has been based on the incidence of human cases. Because of an increasing vaccination rate, this approach might be misleading, especially in regions of low virus circulation. To test the suitability of rodents as a surrogate marker for virus spread, laboratory-bred Microtus arvalis voles were experimentally infected with TBEV and analyzed over a period of 100 days by real-time (RT)-quantitative polymerase chain reaction. Further, the prevalence of TBEV in rodents trapped in Brandenburg, a rural federal state in northeastern Germany with autochthonous TBE cases, was determined and compared with that in rodents from German TBE risk areas as well as TBE nonrisk areas. In experimentally infected M. arvalis voles, TBEV was detectable in different organs for at least 3 months and in blood for 1 month. Ten percent of all rodents investigated were positive for TBEV. However, in TBE risk areas, the infection rate was higher compared with that of areas with only single human cases or of nonrisk areas. TBEV was detected in six rodent species: Apodemus agrarius, Apodemus flavicollis, Apodemus sylvaticus, Microtus agrestis, Microtus arvalis, and Myodes glareolus. M. glareolus showed a high infection rate in all areas investigated. The infection experiments proved that TBEV can be reliably detected in infected M. arvalis voles. These voles developed a persistent TBE infection without clinical symptoms. Further, the study showed that rodents, especially M. glareolus, are promising sentinels particularly in areas of low TBEV circulation.

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

BackgroundTick-borne encephalitis is the most common tick-borne viral infection in Europe with 3,000 human cases reported each year. In Western Europe, the castor bean tick, Ixodes ricinus, is the principal vector of the tick-borne encephalitis virus (TBEV). TBEV appears to be spreading geographically and was recently detected for the first time in Canton Valais in the southern part of Switzerland. The purpose of the present study was to survey the I. ricinus tick populations of Canton Valais for TBEV.MethodsWe collected a total of 19,331 I. ricinus ticks at 45 different sites in Canton Valais between 2010 and 2013. Ticks were processed in pools and tested for TBEV using reverse transcription quantitative PCR. The NS5 gene and the envelope gene of the TBEV isolates were partially sequenced for phylogenetic analysis.ResultsTBEV was detected in tick populations at six of the 45 sites. These six sites were all located in a 33 km transect along the Rhône River. TBEV was detected in two sites for three of the four years of the study showing the temporal persistence of the pathogen. Prevalence of TBEV in the six positive sites ranged from 0.16% to 11.11%. Phylogenetic analysis found that all TBEV isolates from Canton Valais belonged to the European subtype. Genetic analysis found two distinct lineages of TBEV suggesting that Canton Valais experienced two independent colonization events.ConclusionsTBEV appears to be well established at certain locations in Canton Valais.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-3305-7-443) contains supplementary material, which is available to authorized users.