Stankevičius, Arūnas

Several studies have documented reverse zoonotic transmission of SARS-CoV-2, including in farmed mink which are susceptible to human respiratory viruses and are known for serving as a reservoir capable of generating new virus variants in densely populated farms. Here, we present the results of a genomic investigation launched in response to detected human infections with mink-origin SARS-CoV-2 lineages, and show evidence of at least 14 high-confidence introductions of SARS-CoV-2 from humans into farmed mink in Lithuania where sustained transmission in farmed mink lasted up to a year. We estimated the most likely timeframes for these introductions encompassing at least six SARS-CoV-2 lineages, some of which were already extinct in humans, with Bayesian phylogenetic and molecular clock analyses. This study highlights the public health risks posed by fur farms and underscores that passive genomic surveillance systems are ineffective without the active involvement and expertise of responsible institutions.

Tick-borne encephalitis virus (TBEV) is a major neurotropic virus circulating in Lithuania, maintained through a natural cycle between ticks and vertebrate hosts. Although RT-qPCR is widely used for virus detection in field samples, quantitative data on viral replication, particularly from infected cell cultures, remain limited. To investigate this aspect, we analyzed 139 TBEV-positive samples originating from ticks (n = 16), rodents (n = 69), dogs (n = 32), horses (n = 7), goats (n = 4), and raw goat milk (n = 11). Viral RNA copy number per microliter was determined using absolute quantification with digital PCR. Additionally, positive samples were cultured through ten consecutive passages in three different cell lines - MARC145, Vero, and N2a, followed by digital PCR to evaluate their capacity for viral amplification. Mean viral loads measured in native samples (viral copies/µL) were: horse blood 6.14 (95% CI 0 – 12.59), ticks 3.36 (95% CI 1.95 – 4.77), rodent tissues 6.72 (95% CI 3.82 – 9.63), goat blood 2.56 (95% CI 0 – 5.52), goat milk 4.54 (95% CI 2.28 – 6.8), and dog blood 4.25 (95% CI 2.76 – 5.74). Inferential analysis confirmed a statistically significant passage-dependent increase only in MARC-145 cells (slope = 0.20, 95% CI 0.12 – 0.27), (p < 0.001), although overall viral load remained very low. Furthermore, comparison of viral loads before and after cell culture revealed a strong increase only in the vector group (p < 0.001), while the domestic and synanthropic groups did not change significantly. These findings highlight the utility of digital PCR for precise viral quantification in diverse biological matrices, aiding improvement of diagnostic approaches and risk assessment in veterinary and public health contexts. Moreover, despite being rarely used for TBEV culture in practice, MARC-145 cells could be a promising first-choice system for experimental applications. However, due to very low viral copy numbers, amplification in cell culture is not a suitable method for sample preparation when a high viral load is required.

Tick-borne encephalitis virus (TBEV) is a major zoonotic pathogen in Europe, yet its impact on canine health remains underrecognised in clinical veterinary practice. In a recent large-scale study in Lithuania, one of the most highly endemic countries for TBE, we examined serum samples from 473 dogs collected in two Lithuanian veterinary clinics. Using ELISA for serology and RT-PCR targeting the NCR5 region for viral RNA detection, we found that 21.6% (CI 95% 17.9–25.6) of dogs carried TBEV-specific antibodies, while 18.6% (CI 95% 15.2–22.4) were PCR-positive. Neurological signs were observed in 31.8% of PCR-positive animals, with a case fatality/euthanasia rate of 18.2%. Male dogs were more likely to develop neurological manifestations, and older age, neurological signs, and the presence of antibodies were significantly associated with poor outcomes. Infectious virus was successfully propagated in cell culture from PCR-positive sera, confirming active infection. These findings highlight that TBEV should be considered an important differential diagnosis in dogs presenting with acute neurological disease in endemic areas. Awareness and early diagnostic testing are essential for veterinarians to improve case outcomes and to better understand the epidemiology of this canine pathogen.

Climate change is reshaping tick ecology by prolonging their seasonal activity and enabling expansion into previously unsuitable habitats [1]. Consequently, the incidence of tick-borne diseases is increasing in regions once considered low risk, creating growing public health challenges. Tick-borne encephalitis (TBE) remains one of the most important vector-borne diseases in Europe, with the Baltic countries accounting for nearly one-third of reported cases [2, 3]. This study investigated the prevalence of tick-borne encephalitis virus (TBEV) in questing ticks in Lithuania and evaluated key transmission risk factors. During 2023–2024, 544 ticks (Ixodes ricinus, n = 525; Dermacentor reticulatus, n = 19) were collected by flagging method in urban habitats and pooled into 47 groups by species and developmental stage. TBEV detection was carried out using real-time RT-PCR and virus isolation in two cell lines (Neuro-2a and MARC-145). TBEV RNA was detected in 15.26% (83/544) of individual tick samples. Virus isolation proved more sensitive than direct RT-PCR, yielding a total minimum infection rate (MIR) of 2.8% across both cell lines, compared with 1.47% by direct RT-PCR. MIR was 1.3% (7/525) for I. ricinus and 5.3% (1/19) for D. reticulatus. Positive samples included two nymphs, three females, and three males, indicating active TBEV circulation across developmental stages and sexes. These findings highlight a potentially greater role of D. reticulatus in TBEV transmission than previously recognised and emphasise the need for continued tick surveillance, robust public health interventions, and the inclusion of virus isolation techniques to enhance detection accuracy.

Tick-borne encephalitis virus (TBEV) is maintained in complex natural cycles involving multiple hosts and vectors ticks. While TBEV detection in field samples is routinely performed using RT-qPCR, quantitative data on viral load across different origins remain scarce [1]. In this study, we analysed 213 TBEV-positive samples originating from ticks, rodents, goats, dogs, horses, and raw goat milk. Viral RNA copy number per microliter was determined using absolute quantification with digital PCR. Mean viral loads (viral copies/µL) after outlier elimination were: horses 6.68, ticks 3.45, rodents 4.06, goats 3.64, milk 2.80, and dogs 3.89. Statistical comparisons revealed significantly higher viral loads in horse samples compared to ticks (p = 0.0012), rodents (p = 0.0262), goat milk (p = 0.0110), and dogs (p = 0.0398). Without outlier removal, the corresponding means were higher overall: horses 31.35, ticks 8.33, rodents 8.23, goats 3.64, milk 3.57, and dogs 3.89. These findings highlight substantial variation in the TBEV copy number depending on sample origin and wide variation within a sample type. Our results further demonstrate the utility of digital PCR for precise viral quantification in diverse biological matrices, which may aid in refining diagnostic approaches and risk assessment in both veterinary and public health contexts.

Tick-borne encephalitis virus (TBEV) is an emerging concern in canine health [1–2], yet its active circulation in dogs remains insufficiently documented. In a cross-sectional study of 473 dogs from central Lithuania (2020–2021), we confirmed acute infections by PCR, serology, and virus isolation in cell culture. TBEV RNA was detected in 18.6% of canine samples, and antibodies were present in 21.6%. Importantly, we succeeded in isolating infectious TBEV from seven serum samples and one cerebrospinal fluid specimen. Replication was demonstrated in Neuro-2a, Vero, and Marc-145 cells, with subsequent confirmation by sequencing. These results provide direct evidence that viraemic dogs carry transmissible virus and can serve as a source for successful cultivation. In parallel, attached ticks collected from the same dogs carried TBEV RNA in 34.2% of suspensions. After three blind passages in cell culture, positivity rose to 56–61%, highlighting that viral replication in ticks often requires amplification to reach detectable levels. Nevertheless, no consistent concordance was found between a dog’s infection status and TBEV detection in its ticks. The study demonstrates that virus isolation from naturally infected dogs and their ticks is feasible and reliable, confirming the role of dogs as suitable sentinel hosts for TBEV. Cultivation in multiple cell lines expands opportunities for experimental studies and strain characterisation. Our findings underline the clinical and epidemiological importance of canine TBEV infections and provide valuable resources for further virological and pathogenesis research.

Background: Equine piroplasmosis (EP), a tick-borne disease caused by Theileria equi and Babesia caballi, poses a significant health concern for equines worldwide (1, 2). In Lithuania, no previous data have been published regarding the prevalence or diagnostic testing for these pathogens. Given the growing concern over tick populations and climate change, it is critical to assess the current epidemiological status of EP within the country (1). Aim(s): This study aimed to determine the prevalence of T. equi and B. caballi in Lithuanian horses using both serological and molecular diagnostic methods, and to identify potential risk factors associated with the infection. Methods: A total of 152 serum samples and 83 whole blood samples were collected from clinically healthy horses across twelve municipalities in Lithuania. The sampled horses varied in age, sex, and breed. Competitive enzyme-linked immunosorbent assay (cELISA) was used to detect specific antibodies, while polymerase chain reaction (PCR) targeting the 18S rRNA gene was employed to detect parasite DNA. Results: Piroplasm-specific IgG antibodies were detected in 7.89 ± 4.4% of serum samples. PCR revealed T. equi or B. caballi DNA in 27.7 ± 9.5% of blood samples. Antibodies were found in horses from four municipalities, while PCR confirmed infections in horses from six out of seven tested municipalities. The highest seroprevalence was found in Palanga district (46.2%), and the highest PCR prevalence in Trakai district (60.0%). A statistically significant association was observed between infection rates and horse breed, as well as the municipality of residence (p<0.05). Conclusion This research represents the first documentation of the prevalence of Theileria equi and Babesia caballi within the equine population of Lithuania, employing both serological and molecular methodologies. The results highlight the importance of ongoing surveillance using both serological and molecular methods. Given the influence of environmental factors on vector populations, broader nationwide monitoring is recommended to protect equine health.

Climate change has significantly influenced tick ecology by extending their seasonal activity and facilitating their expansion into new habitats. As a result, the incidence of tick-borne diseases is rising in previously low-risk areas, posing increasing public health concerns. Among these, tick-borne encephalitis (TBE) is one of the most significant vector-borne diseases in Europe, with the Baltic countries accounting for nearly one-third of reported European cases. This study assessed the prevalence of tick-borne encephalitis virus (TBEV) in questing ticks in Lithuania and key risk factors for transmission. Between 2023 and 2024, 544 ticks (Ixodes ricinus, n = 525; Dermacentor reticulatus, n = 19) were collected using the flagging method in urban areas and grouped into 47 pools based on species and developmental stages. TBEV detection was performed using real-time RT-PCR and virus isolation in two cell lines (Neuro-2a, MARC-145). TBEV RNA was detected in 15.26% (83/544) of samples. Virus isolation proved more effective than direct RT-PCR, with a total minimum infection rate (MIR) of 4.41% across both cell lines, compared to 1.47% from direct RT-PCR. The MIR for I. ricinus was 1.3% (7/525) and for D. reticulatus 5.3% (1/19). Positive samples included six nymphs, nine females, and seven males, highlighting widespread TBEV circulation across developmental stages and sexes. These findings suggest that D. reticulatus may play a greater role in TBEV transmission than previously recognised. The study underscores the need for ongoing tick surveillance and public health strategies, as well as the importance of virus isolation for improving detection accuracy.

Wild rodents are recognized as key reservoir hosts for tick-borne encephalitis virus (TBEV), making them valuable indicators for identifying TBEV foci. This study aimed to evaluate the efficacy of viral RNA detection in rodents for confirming suspected TBEV hotspots. A total of 139 small rodents were captured from various known TBEV-endemic locations across Lithuania. Rodent samples were cultured in murine neuroblastoma Neuro-2a cells to increase viral RNA detectability. TBEV RNA was detected in 74.8% (95% CI: 66.7–81.1) of brain and internal organ mix suspensions. The prevalence rate significantly increased after sample cultivation in Neuro-2a cells. Notably, a strong correlation (r = 0.88; p < 0.05) was observed between TBEV RNA prevalence after cultivation in cells and the average monthly air temperature during rodent trapping. This study highlights the suitability of wild rodents as sentinel hosts for TBEV surveillance and demonstrates the effectiveness of cell culture methods in amplifying viral load for detection.

Tick-borne encephalitis (TBE) is recognized as the most important zoonotic tick-borne viral disease in Europe. Causative agent of the disease is typically transmitted to human beings through bites of tick-borne encephalitis virus (TBEV)-infected ticks. However, an increasing number of case reports of TBE infection in dogs indicate that the virus might be an important tick-borne pathogen in dogs, especially in endemic areas. Therefore, the aim of the present study was to investigate the prevalence of TBEV RNA and TBEV-specific antibodies in serum samples of dogs living in a highly endemic region of Lithuania and to evaluate the main risk factors associated with the severe cases of TBE. The serum samples (n = 473) of dogs were randomly collected in two veterinary clinics in central Lithuania and analysed by commercially available ELISA kits and nested RT-PCR using primers targeting NCR5 region. To confirm TBEV propagation, Neuro-2a, Vero and MARC-145 cells were transfected with suspensions prepared from positive sera and viral replication in cells was confirmed via RT-PCR. Observed neurological symptoms in 31.8% (CI 95% 22.3-42.6) and a death rate of 18.2% (CI 95% 10.8-27-8) of PCR-positive dogs implies that TBE is a clinically important disease in dogs in endemic countries such as Lithuania. TBEV-specific antibodies were detected in 21.6% (CI 95% 17.9-25.6) of randomly collected dog blood samples and in 32.1% (CI 95% 15.9-52.4) of dogs with neurological symptoms. Males were more likely to develop neurological symptoms (p = 0.0084), while older dogs (p = 0.0032), dogs with neurological symptoms (p = 0.003) and the presence of TBEV-specific antibodies (p = 0.024) were more likely to experience worse outcomes of the disease. The results of the present study demonstrate that TBEV is a common and clinically important pathogen in dogs in Lithuania.