Evaluating the dynamics and efficacy of a live, attenuated Mycoplasma anserisalpingitidis vaccine candidate under farm conditions.

The aim of the present study was to monitor the dynamics and to measure the safety and efficacy of a live, attenuated, thermosensitive Mycoplasma anserisalpingitidis vaccine candidate, namely MA271, in geese breeder flocks under field conditions. Two rearing flocks were vaccinated with MA271 at 4 weeks of age and boosted at 24 weeks of age by cloaca inoculation (1 ml) and eye-dropping (60 µl). The geese then were transported to multi-aged breeding farms. Two breeding flocks served as controls. Colonization of the cloaca by MA271 showed 75% maximum prevalence between 4 and 6 weeks after the first vaccination. Then the prevalence decreased to 25% until the cooler, humid fall months which coincided with the booster vaccination. Boosting raised cloacal colonization to 100%. No clinical signs were observed in the vaccinated birds. After transportation to five multi-aged breeding farms, the wild-type strain appeared as well as MA271 in three flocks. In one flock, the wild-type strain completely displaced MA271, while in one flock only MA271 was detected. Only wild-type strains were detected in the control flocks; however, due to an HPAI outbreak, both flocks were exterminated before the end of the study. Based on the available data, the median percentage of infertile eggs was 3.7-5.1% in the MA271 vaccinated flocks, and 7.7% in the non-vaccinated flock. In conclusion, MA271 can colonize the cloaca of geese under field conditions. MA271 proved to be safe and presumably protects against M. anserisalpingitidis-induced reproduction losses.

Pollution impact assessment of secondary iron smelting on soil and some medicinal herbs grown at Fasina community in Ile-Ife, Nigeria.

Use of medicinal herbs is now gaining popularity especially among the low-income people because it is cheap, readily available and its "seeming" lack of side effects. However, environmental pollution is a potential threat to its continued use. This study examines the effect of air pollution on the soil and consequently on the medicinal herbs grown on such soils. Soil and four medicinal herbs, Chromolaena odorata, Vernonia amygdalina, Carica papaya and Ocimum gratissimum, commonly used in the south western part of Nigeria either as purely medicinal herbs, soup vegetables or both were carefully harvested from Fasina, a polluted area, and Moro, a relatively unpolluted area, all in Ile-Ife, Nigeria. Samples were prepared following standard practice and analysed for nickel, chromium, cadmium and lead using atomic absorption spectroscopy (AAS). The results showed that elemental concentrations at the two locations were within the permissible limit for both soil and herbs, the statistical test also established no significant difference between the two locations. However, toxic metals concentrations (chromium, cadmium and lead) were found higher at the polluted site while that of the essential metal, nickel, was higher at the unpolluted site. Of the four metals, cadmium has the highest transfer ratio (0.39 and 0.34) while lead has the least (0.21 and 0.25) for Moro and Fasina sites respectively. Similarly, Chromolaena odorata has the highest transfer ratio (0.34) while Carica papaya has the least (0.28). In conclusion, gradual build-up of the toxic metals at the polluted site is evident and may eventually contaminate the herbs.

Disentangling the role of poultry farms and wild birds in the spread of highly pathogenic avian influenza virus in Europe.

In winter 2016-7, Europe was severely hit by an unprecedented epidemic of highly pathogenic avian influenza viruses (HPAIVs), causing a significant impact on animal health, wildlife conservation, and livestock economic sustainability. By applying phylodynamic tools to virus sequences collected during the epidemic, we investigated when the first infections occurred, how many infections were unreported, which factors influenced virus spread, and how many spillover events occurred. HPAIV was likely introduced into poultry farms during the autumn, in line with the timing of wild birds' migration. In Germany, Hungary, and Poland, the epidemic was dominated by farm-to-farm transmission, showing that understanding of how farms are connected would greatly help control efforts. In the Czech Republic, the epidemic was dominated by wild bird-to-farm transmission, implying that more sustainable prevention strategies should be developed to reduce HPAIV exposure from wild birds. Inferred transmission parameters will be useful to parameterize predictive models of HPAIV spread. None of the predictors related to live poultry trade, poultry census, and geographic proximity were identified as supportive predictors of HPAIV spread between farms across borders. These results are crucial to better understand HPAIV transmission dynamics at the domestic-wildlife interface with the view to reduce the impact of future epidemics.

First DNA sequence proof for the occurrence of bovine adenovirus types 10 and 11 in continental Europe.

Bovine adenoviruses (BAdV) are known to cause respiratory and/or intestinal disease in calves. Infection can manifest as acute outbreaks, but more often only sporadic cases occur. Here we describe the PCR detection and partial sequence characterization of several BAdVs found in sick or dead calves on different farms in Western Hungary. Intermittent diarrhoeal illnesses occurred after weaning among calves on several farms located up to 40 km apart. A high-sensitivity, broad-spectrum nested PCR, developed for the general detection of adenoviruses, gave positive results in four independent cases. Direct sequencing of PCR products showed clear results from only two samples, whereas sequences from the other two amplicons were mixed. Molecular cloning of these heterogeneous PCR products was performed to separate each DNA fragment therein. By sequencing several plasmid clones from both mixed samples, we were able to detect the simultaneous presence of two different BAdV types, namely types 6 and 10 classified into two separate (Atadenovirus and Mastadenovirus) genera. The sequence of one homogenous sample was identified as being derived also from BAdV-10, whereas the other sample contained a novel type, proposed to be BAdV-11. We demonstrated, for the very first time, the occurrence of the two latter virus types in continental Europe. Their appearance in Hungary marks a significant shift in the types of BAdVs actually circulating in the country. Considering the similarity of the pathological findings to those, attributed to BAdV-10 infections reported to date, the causative role of the viruses in these cases seems to be plausible. Phylogeny reconstruction further confirmed that BAdVs represent multiple genetic lineages.

A universal RT-qPCR assay for "One Health" detection of influenza A viruses.

The mutual dependence of human and animal health is central to the One Health initiative as an integrated strategy for infectious disease control and management. A crucial element of the One Health includes preparation and response to influenza A virus (IAV) threats at the human-animal interface. The IAVs are characterized by extensive genetic variability, they circulate among different hosts and can establish host-specific lineages. The four main hosts are: avian, swine, human and equine, with occasional transmission to other mammalian species. The host diversity is mirrored in the range of the RT-qPCR assays for IAV detection. Different assays are recommended by the responsible health authorities for generic IAV detection in birds, swine or humans. In order to unify IAV monitoring in different hosts and apply the One Health approach, we developed a single RT-qPCR assay for universal detection of all IAVs of all subtypes, species origin and global distribution. The assay design was centred on a highly conserved region of the IAV matrix protein (MP)-segment identified by a comprehensive analysis of 99,353 sequences. The reaction parameters were effectively optimised with efficiency of 93-97% and LOD95% of approximately ten IAV templates per reaction. The assay showed high repeatability, reproducibility and robustness. The extensive in silico evaluation demonstrated high inclusivity, i.e. perfect sequence match in the primers and probe binding regions, established as 94.6% for swine, 98.2% for avian and 100% for human H3N2, pandemic H1N1, as well as other IAV strains, resulting in an overall predicted detection rate of 99% on the analysed dataset. The theoretical predictions were confirmed and extensively validated by collaboration between six veterinary or human diagnostic laboratories on a total of 1970 specimens, of which 1455 were clinical and included a diverse panel of IAV strains.

Genomic Analysis of Staphylococcus aureus Strains Originating from Hungarian Rabbit Farms Reinforce the Clonal Origin of Various Virulence Types.

Staphylococcosis is one of the most important infectious diseases in rabbit medicine, especially in commercial farming. Previous studies revealed the existence of virulent variants adapted to rabbits. Typical and atypical, highly virulent as well as low virulent variants have been isolated and reported from industrial units in all major rabbit-meat-producing countries. Preceding the research focused on detecting defined nucleotide sequences, the genome of these organisms as a whole was rarely subjected to scientific investigations. The authors sequenced 51 Staphylococcus strains originating from industrial rabbit farms in Hungary. Another 12 draft genomes of rabbit isolates were constructed from read sequences available in digital repositories, and were compared based on whole-genome multilocus sequence typing. The clonal origin of highly virulent variants is confirmed, the strains from Hungary were closely related with the strains isolated in the UK, Italy, and Spain. Atypical highly virulent strains are the most prevalent in Hungary, they form a separate clonal cluster. The low virulent strains were genetically similar, but more heterogeneous than the highly virulent (HV) and aHV strains even by the traditional MLST typing scheme. Other "non-aureus" Staphylococcus species were also identified.

Publisher Correction: Occurrence and genetic diversity of CRESS DNA viruses in wild birds: a Hungarian study.

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

Molecular epidemiology and phylodynamics of goose haemorrhagic polyomavirus.

Goose haemorrhagic polyomavirus (GHPV, or Anser anser polyomavirus 1) is a small dsDNA virus of the Polyomaviridae family. The virus infects the internal organs causing haemorrhagic nephritis and enteritis of geese that may be fatal for goslings. In this study, GHPV positivity was examined in goose and duck samples collected in Hungary between 2005 and 2019. In this period, 384 of the investigated 1,111 specimens were diagnosed as GHPV-positive by PCR assay. Twenty-two GHPV genomes were sequenced and subjected to phylogenetic and evolutionary analysis. Based on the sequence data, the mean evolutionary rates were estimated 6.57 × 10-6 -5.82 × 10-5  s/s/y for both GHPV complete genomes and individual genes, with negative selection acting on each gene. When GHPV VP1 sequences originating from wild birds were also included in the analyses, the nt and aa mutations inflated the substitution rate to 1.54 × 10-4  s/s/y that may imply adaptation of the virus to novel host species. Our data suggested the co-circulation of various GHPV strains in Hungarian goose farms; the source of these may be persistently infected domesticated or migratory wild birds. Detection and characterization of GHPV in wild birds and domestic waterfowls may help to elaborate new strategies for more effective disease control and prevention.

Low incidence of Schmallenberg virus infection in natural cases of abortion in domestic ruminants in Hungary.

An epizootic caused by a new orthobunyavirus called Schmallenberg virus (SBV) was recognised in European ruminants in 2011 and 2012. The re-emergence of the infection was reported in several countries in the subsequent years. Although the main clinical sign of SBV infection is abortion, the impact of SBV in natural cases of abortion in domestic ruminants had not been systematically examined before this study. The aim of the study was to investigate the role of SBV infection and to compare it to the importance of other causes of abortion by examining 537 natural cases of abortion that had occurred between 2011 and 2017 in Hungary. The cause of abortion was determined in 165 (31%) cases. An infectious cause was proved in 88 (16%) cases. SBV infection was found only in a total of four cases (0.8%) using real-time polymerase chain reaction. Three of them proved to be inapparent SBV infection, and one case was attributed to SBV-induced abortion by detecting non-purulent encephalitis and SBV nucleoprotein by immunohistochemistry in a brain tissue sample. According to the results, SBV played a minor role in natural cases of domestic ruminant abortion in Hungary during the 7-year period following the first SBV outbreak in 2011.

Occurrence and genetic diversity of CRESS DNA viruses in wild birds: a Hungarian study.

Circoviruses, cycloviruses and other circular, replication-associated protein-encoding single stranded (CRESS) DNA viruses have been detected in a variety of animal taxa. In this study, cloacal swab samples (n = 90) were examined for CRESS DNA viruses from 31 wild bird species living at various aquatic sites in Hungary to identify possible reservoirs of viruses pathogenic to domestic poultry. A total of 30 (33.3%) specimens tested positive with pan-CRESS DNA virus specific PCR. Goose circovirus (GoCV), Duck associated cyclovirus 1 (DuACyV-1) and Garrulus glandarius associated circular virus 1 (GgaCV-1) were detected in nine, three and two different bird species, respectively. Selected specimens were subjected to whole genome sequencing. The obtained sequence data revealed conserved gene structure within the identified virus species and detected homologous (within GoCV) and possible heterologous recombination (within DuACyV-1) events. Results presented here provide new information on the genomic diversity and evolution of selected CRESS DNA viruses.

Isolation and characterisation of porcine epidemic diarrhoea virus in Hungary - Short communication.

Porcine epidemic diarrhoea virus (PEDV) is an emerging enteropathogen, causing great economic losses in the pig industry. After many years of quiescence, PEDV was detected in Hungary in 2016 with a recombination in its S gene. In order to determine the extent of this change, an attempt was made to isolate the recombinant PEDV. This study was extended with a variety of samples collected from three separate farms with newly identified PEDV in 2018. The recombinant PEDV from 2016 was isolated successfully along with three viruses from 2018, and one isolate from the new cases was used for whole genome determination. Whole genome sequence alignment revealed the highest identity with recombinant Hungarian and Slovenian PEDV within the low-pathogenic European viruses. This suggests that these recombinant PEDV are circulating in this area and may spread to other parts of the continent.

Virus-Like Particle Based Vaccine Provides High Level of Protection Against Homologous H5N8 HPAIV Challenge in Mule and Pekin Duck, Including Prevention of Transmission.

The most recent pandemic clade of highly pathogenic avian influenza (HPAI) H5, clade 2.3.4.4, spread widely, with the involvement of wild birds, most importantly wild waterfowl, carrying the virus (even asymptomatically) from Asia to North America, Europe, and Africa. Domestic waterfowl being in regular contact with wild birds played a significant role in the H5Nx epizootics. Therefore, protection of domestic waterfowl from H5Nx avian influenza infection would likely cut the transmission chain of these viruses and greatly enhance efforts to control and prevent disease outbreak in other poultry and animal species, as well as infection of humans. The expectation for such a vaccine is not only to provide clinical protection, but also to control challenge virus transmission efficiently and ensure that the ability to differentiate infected from vaccinated animals is retained. A water-in-oil emulsion virus-like particle vaccine, containing homologous hemagglutinin antigen to the current European H5N8 field strains, has been developed to meet these requirements. The vaccine was tested in commercial Pekin and mule ducks by vaccinating them either once, at 3 wk of age, or twice (at 1 day and at 3 wk of age). Challenge was performed at 6 wk of age with a Hungarian HPAIV H5N8 isolate (2.3.4.4 Group B). Efficacy of vaccination was evaluated on the basis of clinical signs, amount of virus shedding, and transmission. Vaccination resulted in complete clinical protection and prevention of challenge virus transmission from the directly challenged vaccinated ducks to the vaccinated contact animals.

Una vacuna basada en partículas similares a virus proporciona un alto nivel de protección contra el desafío con un virus homólogo de influenza aviar de alta patogenicidad H5N8 en patos mula y Pekin, incluida la prevención de la transmisión. El clado pandémico más reciente de influenza aviar altamente patógena H5, clado 2.3.4.4, se diseminó ampliamente, con la participación de aves silvestres, siendo las aves acuáticas más importantes, portando el virus (incluso asintomáticamente) de Asia a América del Norte, Europa, y África. Las aves acuáticas domésticas en contacto regular con aves silvestres desempeñaron un papel importante en las epizootias H5Nx. Por lo tanto, la protección de las aves acuáticas domésticas contra la infección por influenza aviar H5Nx probablemente cortaría la cadena de transmisión de estos virus y aumentaría en gran medida los esfuerzos para controlar y prevenir brotes de enfermedades en otras aves comerciales y especies animales, así como la infección en humanos. La expectativa de una vacuna de este tipo es no solo brindar protección clínica, sino también controlar la transmisión del virus de desafío de manera eficiente y garantizar que se mantenga la capacidad de diferenciar a los animales vacunados. Se ha desarrollado una vacuna emulsionada en aceite con partículas similares al virus, que contiene el antígeno de hemaglutinina homóloga a las cepas de campo H5N8 europeas actuales, para cumplir con estos requisitos. La vacuna se probó en patos de Pekín y mulas comerciales, vacunándolos una vez, a las tres semanas de edad, o dos veces (al primer día y a las tres semanas de edad). El desafío se realizó a las seis semanas de edad con un aislado de alta patogenicidad H5N8 húngaro (2.3.4.4 Grupo B). La eficacia de la vacunación se evaluó en función de los signos clínicos, la eliminación viral y la transmisión. La vacunación dio como resultado una protección clínica completa y la prevención de la transmisión del virus de desafío de los patos vacunados.

Co-circulation of genetically distinct highly pathogenic avian influenza A clade 2.3.4.4 (H5N6) viruses in wild waterfowl and poultry in Europe and East Asia, 2017-18.

Highly pathogenic avian influenza (HPAI) H5 clade 2.3.4.4 viruses were first introduced into Europe in late 2014 and re-introduced in late 2016, following detections in Asia and Russia. In contrast to the 2014-15 H5N8 wave, there was substantial local virus amplification in wild birds in Europe in 2016-17 and associated wild bird mortality, with evidence for occasional gene exchange with low pathogenic avian influenza (LPAI) viruses. Since December 2017, several European countries have again reported events or outbreaks with HPAI H5N6 reassortant viruses in both wild birds and poultry, respectively. Previous phylogenetic studies have shown that the two earliest incursions of HPAI H5N8 viruses originated in Southeast Asia and subsequently spread to Europe. In contrast, this study indicates that recent HPAI H5N6 viruses evolved from the H5N8 2016-17 viruses during 2017 by reassortment of a European HPAI H5N8 virus and wild host reservoir LPAI viruses. The genetic and phenotypic differences between these outbreaks and the continuing detections of HPAI viruses in Europe are a cause of concern for both animal and human health. The current co-circulation of potentially zoonotic HPAI and LPAI virus strains in Asia warrants the determination of drivers responsible for the global spread of Asian lineage viruses and the potential threat they pose to public health.

Frequency of diarrhoea-associated viruses in swine of various ages in Hungary.

Enteric viral diseases of swine are one of the most frequent disorders causing huge economic losses in pork production. After the reappearance of an emerging enteropathogen, porcine epidemic diarrhoea virus (PEDV) in Hungary in 2016, an extensive survey was initiated in an attempt to identify diarrhoea-related porcine viruses, including adeno-, astro-, boca-, calici-, circo-, corona-, kobu-, rota- and Torque teno viruses. A total of 384 faecal samples collected during a twoyear period from diarrhoeic and asymptomatic pigs of various ages in 17 farms were screened by conventional and real-time PCR methods. Half of the samples contained at least one examined virus with the dominance of kobuvirus (55.1%) followed by bocaviruses (33.2%) and rotavirus groups A and C together (20.9%), while coronaviruses including PEDV were not found in this set of samples. Statistical analysis showed a highly significant difference (P < 0.0001) in the frequency of single infections compared to mixed ones with the exception of weaned pigs, in which group additionally most viruses were detected. The results of this study suggest that the complexity of this disease may vary with age, which makes the prevention of diarrhoea a challenge, especially in weaned pigs.

Efficacy of a Recombinant Turkey Herpesvirus AI (H5) Vaccine in Preventing Transmission of Heterologous Highly Pathogenic H5N8 Clade 2.3.4.4b Challenge Virus in Commercial Broilers and Layer Pullets.

Outbreaks caused by the highly pathogenic avian influenza virus (HPAIV) H5N8 subtype clade 2.3.4.4 were first reported in 2014 in South Korea then spread very rapidly in Asia, to Europe, and for the first time, to North America. Efficacy of a recombinant HVT-AI (H5) vaccine (rHVT-H5) to provide clinical protection as well as to significantly reduce the shedding of an H5N8 challenge virus has already been demonstrated in SPF chickens. The aim of our studies was to test the efficacy of the same rHVT-H5 vaccine in controlling the transmission of a recent Hungarian HPAIV H5N8 challenge virus in commercial chickens. Broilers and layers were vaccinated at day old according to the manufacturer's recommendation and then challenged with a 2017 Hungarian HPAIV H5N8 (2.3.4.4b) isolate at 5 or 7 weeks of age, respectively. Evaluation of clinical protection, reduction of challenge virus shedding, and transmission to vaccinated contact birds was done on the basis of clinical signs/mortality, detection, and quantitation of challenge virus in oronasal and cloacal swabs (regularly between 1 and 14 days postchallenge). Measurement of seroconversion to AIV nucleoprotein was used as an indicator of infection and replication of challenge virus. Our results demonstrated that rHVT-H5 vaccination could prevent the development of clinical disease and suppress shedding very efficiently, resulting in the lack of challenge virus transmission to vaccinated contact chickens, regardless the type of birds. Single immunization with the tested rHVT-H5 vaccine proved to be effective to stop HPAIV H5N8 (2.3.4.4b) transmission within vaccinated poultry population under experimental conditions.

Comparison of 2016-17 and Previous Epizootics of Highly Pathogenic Avian Influenza H5 Guangdong Lineage in Europe.

We analyzed the highly pathogenic avian influenza (HPAI) H5 epizootic of 2016-17 in Europe by epidemiologic and genetic characteristics and compared it with 2 previous epizootics caused by the same H5 Guangdong lineage. The 2016-17 epizootic was the largest in Europe by number of countries and farms affected and greatest diversity of wild birds infected. We observed significant differences among the 3 epizootics regarding region affected, epidemic curve, seasonality, and outbreak duration, making it difficult to predict future HPAI epizootics. However, we know that in 2005-06 and 2016-17 the initial peak of wild bird detections preceded the peak of poultry outbreaks within Europe. Phylogenetic analysis of 2016-17 viruses indicates 2 main pathways into Europe. Our findings highlight the need for global surveillance of viral changes to inform disease preparedness, detection, and control.

Susceptibility-related differences in the quantity of developmental stages of Myxobolus spp. (Myxozoa) in fish blood.

Here, we investigated the early development of two closely related myxozoan parasites, the highly pathogenic Myxobolus cerebralis, the causative agent of the whirling disease in salmonids, and Myxobolus pseudodispar, a common, non-pathogenic parasite of cyprinids. The aim of our study was to examine under in vivo laboratory conditions whether fish blood is involved in the intrapiscine development of the two parasite species and investigate if there is dissimilarity between the parasite infection intensity in blood and if it varies in terms of host susceptibility and parasite pathogenicity. Highly susceptible, less susceptible and non-susceptible hosts were involved. Blood samples were taken 1 day, 1 week and 1 month post exposure to M. cerebralis and M. pseudodispar, respectively. The prevalence and infection intensity was estimated by parasite-specific quantitative real-time PCR. Although previous findings assumed that M. cerebralis might escape from host immune system by migrating via peripheral nerves, our experimental results demonstrated that M. cerebralis is present in blood during the early stage of intrapiscine development. For the non-pathogenic M. pseudodispar, the highest infection prevalence was found in the original host, common roach Rutilus rutilus, whereas the highest infection intensity was detected in rudd Scardinius erythrophthalmus, a "dead-end" host of the parasite. The presence of M. pseudodispar developmental stages in the blood of both susceptible and non-susceptible cyprinids suggests that the susceptibility differences remain hidden during the early stage of infection. Our findings supply further evidence that host specificity is not determined during the early, intrapiscine development involving the vascular system. Furthermore, we found remarkable differences in the infection dynamics of the two parasite species examined, possibly due to their distinct pathogenicity or variations in adaptive capabilities to immune components in host blood.

Correction: MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay.

[This corrects the article DOI: 10.1371/journal.pone.0151204.].

Microevolution and independent incursions as main forces shaping H5 Hemagglutinin diversity during a H5N8/H5N5 highly pathogenic avian influenza outbreak in Czech Republic in 2017.

Here, we present a comprehensive analysis of the H5N8/H5N5 highly pathogenic avian influenza (HPAI) virus strains detected in the Czech Republic during an outbreak in 2017. Network analysis of the H5 Hemagglutinin (HA) from 99% of the outbreak localities suggested that the diversity of the Czech H5N8/H5N5 viruses was influenced by two basic forces: local microevolution and independent incursions. The geographical occurrence of the central node H5 HA sequences revealed three eco-regions, which apparently played an important role in the origin and further spread of the local H5N8/HPAI variants across the country. A plausible explanation for the observed pattern of diversity is also provided.

Hungarian tick-borne encephalitis viruses isolated from a 0.5-ha focus are closely related to Finnish strains.

Four tick-borne encephalitis virus strains were isolated from a small 0.5-ha focus over a six-year-long period (2011-2016) in Hungary. Two strains with identical genomes were isolated from Ixodes ricinus and Haemaphysalis concinna two months apart, which shows that the virus had not evolved separately in these tick species. Whole-genome sequencing of the virus revealed that the isolates differed from each other in 4 amino acids and 9 nucleotides. The calculated substitution rates indicated that the speed of genome evolution differs from habitat to habitat, and continuously changes even within the same focus. The amino acid changes affected the capsid, envelope, NS2a and NS5 genes, and one mutation each occurred in the 5' and 3' NCR as well as the premembrane, NS2a and NS5 genes. Phylogenetic analyses based on complete coding ORF sequences showed that the isolates belong to the European subtype of the virus and are closely related to the Finnish Kumlinge strains, the Bavarian isolate Leila and two isolates of Russian origin, but more distantly related to viruses from the neighbouring Central European countries. These isolates obviously have a common origin and are probably connected by migrating birds. These are the first published complete Hungarian TBEV sequences.