Evelina Simkute

Wild rodents are considered to be one of the most important TBEV-amplifying reservoir hosts; therefore, they may be suitable for foci detection studies. To investigate the effectiveness of viral RNA detection in wild rodents for suspected TBEV foci confirmation, we trapped small rodents (n = 139) in various locations in Lithuania where TBEV was previously detected in questing ticks. Murine neuroblastoma Neuro-2a cells were inoculated with each rodent sample to maximize the chances of detecting viral RNA in rodent samples. TBEV RNA was detected in 74.8% (CI 95% 66.7–81.1) of the brain and/or internal organ mix suspensions, and the prevalence rate increased significantly following sample cultivation in Neuro-2a cells. Moreover, a strong correlation (r = 0.88; p < 0.05) was found between the average monthly air temperature of rodent trapping and the TBEV RNA prevalence rate in cell culture isolates of rodent suspensions, which were PCR-negative before cultivation in cell culture. This study shows that wild rodents are suitable sentinel animals to confirm TBEV foci. In addition, the study results demonstrate that sample cultivation in cell culture is a highly efficient method for increasing TBEV viral load to detectable quantities.

The rising awareness and increasing number of case reports of tick-borne encephalitis (TBE) 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 rate of TBEV RNA and TBEV-specific antibodies in clinical samples of dogs living in a highly endemic region of Lithuania and to evaluate the main risk factors for severe disease course and death. The blood samples (n = 473) of dogs were collected in two veterinary clinics in central Lithuania. Tick-borne encephalitis virus (TBEV) RNA was detected in 18.6% (88/473; CI 95% 15.2–22.4) and TBEV-specific antibodies were found in 21.6% (102/473; CI 95% 17.9–25.6) of dog blood serum samples after confirmation with a virus neutralization test. The death/euthanasia rate was 18.2% (16/88; CI 95% 10.8–27.8) in PCR-positive dogs. Male dogs were more likely to develop neurological symptoms (p = 0.008). Older dogs (p = 0.003), dogs with the presence of neurological symptoms (p = 0.003), and dogs with 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 such endemic countries as Lithuania.

A reliable surveillance strategy of tick-borne encephalitis virus (TBEV) is necessary to ensure adequate disease control measures. However, current approaches assessing geographical TBEV circulation are ineffective or have significant limitations. In this study we investigated a total of 1363 goat and 312 sheep bulk tank milk samples for the presence of TBEV. Samples were collected from systematically selected farms in Lithuania every 4–5 days from April to November in 2018 and 2019. To validate results, we additionally tested 2685 questing ticks collected in the vicinity of milk collection sites. We found 4.25% (95% CI 3.25–5.47) and 4.48% (95% CI 2.47–7.41) goat and sheep milk samples to be positive for TBEV, respectively. Furthermore, geographical distribution of TBEV in milk samples coincided with the known TBE endemic zone and was correlated with incidence of TBE in humans in 2019. When sampling time coincides, TBEV detection in milk samples is as good a method as via flagged ticks, however bulk milk samples can be easier to obtain more frequently and regularly than tick samples. The minimal infectious rate (MIR) in ticks was 0.34% (CI 95% 0.15–0.64). Therefore, our results confirm that testing milk serves as a valuable tool to investigate the spatial distribution of TBEV at higher resolution and lower cost.

Seroprevalence rates and molecular characterization of hepatitis E virus (HEV) prevalent in the Lithuanian human population has not yet been evaluated. Immunosuppressed individuals have been recognized as a risk group for chronic hepatitis due to HEV genotype 3 (HEV-3) infections. The objectives of the present study were to determine prevalence rates of anti-HEV antibodies among inflammatory bowel disease (IBD) patients and solid organ transplant (SOT) recipients, to isolate and characterize HEV strain present in the Lithuanian human population, and to investigate its capacity to infect non-human primate (MARC-145 and Vero), swine (PK-15) and murine (Neuro-2a) cells in vitro. In the present study, the significant difference of anti-HEV IgG prevalence between healthy (3.0% (95% CI 0–6.3)) and immunosuppressed individuals (12.0% [95% CI 8.1–15.9]) was described. Moreover, our findings showed that anti-HEV IgG seropositivity can be significantly predicted by increasing age (OR = 1.032, p < 0.01), diagnosis of IBD (OR = 4.541, p < 0.01) and reception of SOT (OR = 4.042, <0.05). Locally isolated HEV strain clustered within genotype 3i subtype of genotype 3 and was capable of infecting MARC-145 cells. This study demonstrates higher HEV seroprevalence in the risk group compared to healthy control individuals without confidence interval overlap. The high level of genetic homology between human and animal strains in Lithuania and the capacity of locally isolated strains to infect cells of non-human origin suggests its potential for zoonotic transmission.

Various animal species have been evaluated in depth for their potential as Tick-borne encephalitis virus (TBEV) sentinel species, although evidence for equine capacity is incomplete. Therefore, a comprehensive cross-sectional stratified serosurvey and PCR analysis of selected horses (n = 301) were performed in TBEV endemic localities in Lithuania. Attached and moving ticks (n = 241) have been collected from aforementioned hosts to evaluate natural infectivity of TBEV vectors (Ixodes spp.) in the recreational environments surrounding equestrian centers. All samples were screened for TBEV IgG and positive samples were confirmed by virus neutralization test (VNT). 113 (37.5%) horses from all counties of Lithuania tested positive for TBEV IgG, revealing age and sex indifferent results of equine seroprevalence that were significantly dependent on pedigree: horses of mixed breed were more susceptible to infection possibly due to their management practices. TBEV prevalence in equine species corresponded to TBEV-confirmed human cases in the precedent year. As much as 3.9% of horses were viraemic with TBEV-RNA with subsequent confirmation of TBEV European subtype. 4/38 of tested tick pools were positive for TBEV-RNA (Minimal infectious rate 1.2%). Several unknown microfoci were revealed during the study indicating areas of extreme risk close to popular human entertainment sites. The study provides important evidence in favor of horses’ usage as sentinel species, as equines could provide more detailed epidemiological mapping of TBEV, as well as more efficient collection of ticks for surveillance studies.

Wild boar-derived hepatitis E (HEV) genotype 3 virus has been successfully isolated in cell lines of human origin only. Considering the zoonotic potential and possible extrahepatic localisation of genotype 3 strain, it is important to investigate the viability of cell lines of different animal and tissue origins. Therefore, the objective of the present study was to determine the permissiveness of non-human primate (MARC-145 and Vero) and swine (PK-15) cell lines of kidney origin, and a mouse neuroblastoma (Neuro-2a) cell line for isolation of wild boar-derived HEV genotype 3. This study showed that MARC-145, PK-15, Neuro-2a and Vero cell lines were permissive to wild boar-derived HEV genotype 3 subtype 3i harbouring viral genome equivalents of 1.12 × 10⁷ copies/ml, 2.38 × 10⁵ copies/ml, 2.97 × 10⁷ copies/ml and 4.01 × 10⁷ copies/ml after five serial passages respectively. In all permissive cell lines, HEV was continuously recovered from growth medium between five and at least 28 days post-infection. Peak loads of HEV genome equivalents were observed on days 7, 12, 19 and 30 in MARC-145 (2.88 × 10⁷ copies/ml), Vero (4.23 × 10⁶ copies/ml), Neuro-2a (3.15 × 10⁶ copies/ml) and PK-15 (2.24 × 10⁷ copies/ml) cell lines respectively. In addition, successful virus isolation was confirmed by immunofluorescence assay targeting HEV capsid protein and sequencing of HEV isolate retrieved from cell cultures. This study showed that wild boar-derived HEV genotype 3 subtype 3i strain was capable of infecting cell lines of animal origin, including primate and porcine kidney cells (MARC-145, PK-15 and Vero), and mouse neuroblastoma cells (Neuro-2a), supporting the notion of the capacity of HEV genotype 3 to cross the species barrier and extra-hepatic localisation of the virus. These findings warrant further studies of tested cell lines to investigate their capacity as an efficient system for HEV propagation. HEV isolates from other wild animal hosts should be isolated on tested cell lines in order to generate more data on HEV transmission between wild animal populations and their role as sources of human infections.