BACKGROUND West Nile virus (WNV) has become established across Europe, with Hungary serving as a key transmission hub since 2004. Following reduced activity during 2020–22, the 2024 season marked a resurgence with the largest geographical distribution ever recorded in Europe. AIM To analyse the 2024 WNV transmission season in Hungary using a One Health approach and characterise circulating strains within the European phylogeographic context using comprehensive genomic surveillance. METHODS Complete and near-complete genome sequencing was performed on 55 specimens from 38 humans, 15 birds and two Culex pipiens mosquito pools using amplicon-based next-generation sequencing. Phylogeographic analysis incorporated 637 European WNV genome sequences (2004–24) with time-scaled Bayesian phylogenetic reconstruction and continuous spatial diffusion modelling. RESULTS Hungary reported 113 human WNV cases in 2024 (n = 111 autochthonous, 2 imported), a 3.7-fold increase from 2023 (incidence: 1.16 vs 0.31 per 100,000 population). Neuroinvasive disease predominated (92%, n = 104) with a 7.9% case fatality rate. All 55 sequenced strains belonged to WNV lineage 2. Phylogeographic analysis revealed Hungary's central role in European WNV dissemination since 2004, with multiple introductions and local diversification across distinct clades. Continuous spatial modelling identified Hungary as a persistent transmission hub with bidirectional viral flow to neighbouring countries, contributing to northward expansion. CONCLUSION Hungary remains a critical WNV transmission hub in Central Europe with established endemicity of multiple lineage 2 clades. The analysis highlights Hungary's role as both a recipient and major source of European WNV diversity, emphasising the need for coordinated surveillance and climate-adapted preparedness strategies.

Magyarországon jelenleg három lovakat fertőzni képes orthoflavivírus endémiás. A szerológiai módszereken fellépő keresztreakciók sokszor megnehezítik az eredmények értékelését, ezáltal a fertőzések igazolását, amely részben hozzájárulhat a hazánkban évről-évre előforduló szezonális idegrendszeri tüneteket mutató lovak diagnosztizálatlan eseteihez. Az összefoglaló előző két részében a szerzők részletesen ismertették a nyugat-nílusi vírusra adott immunválaszt. Ebben a fejezetben az orthoflavivírus fertőzések diagnosztikájában alkalmazott szerológiai módszerek előnyei, ill. hátrányai kerülnek ismertetésre a kórokozók közötti keresztreakciók tekintetében.

ABSTRACT Background In the last three decades, West Nile virus (WNV, Flaviviridae, Orthoflavivirus genus) has become one of the most important encephalitic agents worldwide, causing substantial numbers of cases in humans and horses every year by re‐emerging in endemic areas and emerging in new territories. It is considered that after natural WNV infection, humans and birds develop long‐term immunoprotection, but data on immunoprotection in horses is scarce. Hypothesis West Nile virus infection provides long‐term humoral immunity in subclinically infected horses. Animals Client‐owned, naturally WNV subclinically infected non‐WNV‐vaccinated, healthy horses. Methods In this prospective cohort study, anti‐WNV neutralizing antibody (nAb) titers of 25 horses were monitored for 5 consecutive years in Hungary. Serum samples were collected annually. First, a WNV immunoglobulin G (IgG) ELISA was performed, followed by virus neutralization tests (VNT) for endemic orthoflaviviruses. A VNT titer > 8 was considered positive. Results The mean WNV titer of horses was 260.64 ± 336.74 in 2019, 114.32 ± 107.36 in 2020, 95.38 ± 115.56 in 2021, 22.53 ± 25.71 in 2022 and 6.31 ± 5.15 in 2023. A significant decrease (p < 0.001) in the nAb titers occurred over time. In 2023, 88% of the horses had WNV VNT titers below the cut‐off value. Conclusion and Clinical Relevance Our results showed a significant decrease in WNV titers over time. Because nAbs correlate best with orthoflavivirus protection, our findings suggest that horses might not be protected against re‐infection. We recommend regular nAb titer testing or vaccination in endemic areas.

West Nile virus (WNV) is a mosquito-borne neurotropic virus that causes neurologic disease in both humans and horses. Yet the long-term cellular immune response following natural infection in horses remains poorly understood. This study aims to evaluate the WNV-specific T-cell response in horses recovered from West Nile neuroinvasive disease (WNND). Twelve client-owned horses (4 Hungarian sport horses, 2 Lippizaners, 1 KWPN, 1 Shagya Arabian, 1 Friesian, 1 Gidran, 1 Andalusian, and 1 draft cross horse) with confirmed clinical WNV infection were enrolled, and peripheral blood mononuclear cells (PBMCs) were collected approximately 290 days post infection. An equine interferon-gamma (IFNγ) Enzyme-Linked Immunospot (ELISpot) assay was performed using a WNV capsid peptide pool as an antigen to assess virus-specific cellular immunity. Results: Ten of twelve horses (83%) exhibited a significant IFNγ response. Statistical analyses revealed no association between ELISpot responses and clinical severity, age, sex, breed, or neutralizing antibody titers. These results demonstrate that naturally infected horses are capable of mounting robust WNV-specific T-cell responses independent of humoral immunity. The findings support a potentially important role for cellular immune memory in long-term protection against WNV reinfection and suggest that the capsid peptide-based ELISpot assay may serve as a useful diagnostic or research tool for the evaluation of orthoflavivirus immunity in equines.

A szerzők szakirodalmi adatok alapján bemutatják a nyugat-nílusi vírus által okozott fertőzésre adott immunválasz legfontosabb elemeit. A nyugat-nílusi vírus (Orthoflavivirus nilense – West Nile virus – WNV) az Orthoflavivirus nemzetségbe sorolt szúnyog terjesztette pozitív szimpla szálú RNS-vírus. A kórokozó minden évben jelentős számú idegrendszeri megbetegedést okoz emberekben és lovakban, emiatt napjainkban az egyik legfontosabb, agyvelőgyulladást okozó arbovírusként tartják számon világszerte. A vírus széleskörű előfordulása és az általa jelentett növekvő fenyegetés ellenére is számos kérdés áll fenn a fertőzések klinikai manifesztációjának tekintetében.

Az előző összefoglalóban a szerzők részletekbe menően ismertették a nyugat-nílusi vírus fertőzésre adott veleszületett immunválasz, valamint az adaptív immunreakció celluláris komponensének folyamatát, ill. ezek szerepét a kórokozó elleni védelemben, adott esetben az immunpatológiás folyamatok kialakulásában. A második rész, a szerezett immunválasz másik ágáról, a humorális immunreakció részleteiről, valamint ennek diagnosztikai és vakcinázási vonatkozásairól nyújt részletes áttekintést, amelyek ismerete a diagnózis felállítása mellett a kórokozó elleni hatékony védekezésben is elengedhetetlen.

West Nile virus (WNV) belongs to the Orthoflavivirus genus within the Flaviviridae family. WNV causes significant number of equine and human neurological cases worldwide. As we discussed in our latest summary, the innate and cellular immune response play substantial role in the protection against the pathogen, however, an exacerbated immune response can lead to immunpathological processses. It is considered that lifelong, or at least long-term immune protection develops in survivors against reinfection. The level of neutralising antibodies is considered the best indicator of the protection against orthoflaviviruses. The envelop protein (E-protein) is the major target of virus neutralising antibodies (nAbs). There is a different magnitude of antigenic similarity between the E-proteins of orthoflaviviruses. Therefore, during an immune response against an orthoflavivirus, not just virus-specific but other orthoflavivirus-specific antibodies are produced, as well. On the one hand, these antibodies can provide protection or enhance the severity of the infection with a heterologous Orthoflavivirus. On the other hand, this phenomenon can make the diagnosis of West Nile virus infection quite difficult in countries with other co-circulating Orthoflaviviruses. Due to the short-term and low titer viraemia in humans and horses, the diagnosis of WNV infection relies on serological methods, however, as stated earlier the interpretation of these results can be difficult due to the cross-reactivity among Orthoflaviviruses. Currently, there are 3 available WNV vaccines for horses in the European Union. All these vaccines guarantee protection against the severe neurological form of the infection for 6-12 months. However, the vaccination protocol for foals and older horses might need to be reconsidered. The second part of our review provides a deep summary of the humoral immune response against WNV with a special focus on the diagnosis and the preventive countermeasures against the infection.

West Nile virus (WNV) is an emerging pathogen in Hungary, causing severe outbreaks in equines and humans since 2007. The aim of our study was to provide a comprehensive report on the clinical signs of West Nile neuroinvasive disease (WNND) in horses in Hungary. Clinical details of 124 confirmed equine WNND cases were collected between 2007 and 2019. Data about the seasonal and geographical presentation, demographic data, clinical signs, treatment protocols, and disease progression were evaluated. Starting from an initial case originating from the area of possible virus introduction by migratory birds, the whole country became endemic with WNV over the subsequent 12 years. The transmission season did not expand significantly during the data collection period, but vaccination protocols should be always reviewed according to the recent observations. There was not any considerable relationship between the occurrence of WNND and age, breed, or gender. Ataxia was by far the most common neurologic sign related to the disease, but weakness, behavioral changes, and muscle fasciculation appeared frequently. Apart from recumbency combined with inappetence, no other clinical sign or treatment regime correlated with survival. The survival rate showed a moderate increase throughout the years, possibly due to the increased awareness of practitioners.

The West Nile virus is endemic in multiple European countries and responsible for several epidemics throughout the European region. Its evolution into local or even widespread epidemics is driven by multiple factors from genetic diversification of the virus to environmental conditions. The year of 2018 was characterized by an extraordinary increase in human and animal cases in the Central-Eastern European region, including Hungary. In a collaborative effort, we summarized and analyzed the genetic and serologic data of WNV infections from multiple Hungarian public health institutions, universities, and private organizations. We compared human and veterinary serologic data, along with NS5 and NS3 gene sequence data through 2018. Wild birds were excellent indicator species for WNV circulation in each year. Our efforts resulted in documenting the presence of multiple phylogenetic subclades with Balkans and Western-European progenitor sequences of WNV circulating among human and animal populations in Hungary prior to and during the 2018 epidemic. Supported by our sequence and phylogenetic data, the epidemic of 2018 was not caused by recently introduced WNV strains. Unfortunately, Hungary has no country-wide integrated surveillance system which would enable the analysis of related conditions and provide a comprehensive epidemiological picture. The One Health approach, involving multiple institutions and experts, should be implemented in order to fully understand ecological background factors driving the evolution of future epidemics.

Five different compounds were tested for their in vitro inhibitory effect against RABV multiplication in mouse neuroblastoma (N2A) cell line. N2A cells were infected with the fixed RABV strain CVS-11 one hour prior to adding antivirals or their respective combinations. The infectious titre of RABV as well as the quantity of viral RNA was determined in the cell culturing medium after 48 h. All five tested compounds (mouse interferon (IFN)-α and -β, ribavirin, favipiravir (T-705) and sorafenib) reduced viral replication in a concentration-dependent manner: IFN-β and sorafenib both provided 73.71% relative inhibition of viral replication in the highest non-cytotoxic concentration, while ribavirin caused 48.07%, IFN-α caused 44.87% and favipiravir caused 35.25% relative inhibition, respectively. When applied in combination, their antiviral activity was not synergistic, but a pronounced inhibition was detected when IFN-β was combined with sorafenib, ribavirin, or favipiravir. The highest antiviral effect was caused by the combination of IFN-β and sorafenib (77.19% relative inhibition). In other combinations there was an antagonistic effect detected in the reduction of viral replication. The results demonstrate that these compounds can be promising candidates for a potential combination treatment of rabies, noting that some combinations are not favourable in vitro, which makes thorough in vivo studies necessary.

A serosurvey for Tahyna virus (TAHV), a mosquito-borne California encephalitis orthobunyavirus (Peribunyaviridae) endemic to Europe, was performed to estimate the activity of TAHV on a broad geographic scale. Sera from wild boar (Sus scrofa), roe deer (Capreolus capreolus) and red deer (Cervus elaphus) were collected from Austria, Hungary and Romania. Samples were tested for neutralizing antibodies against TAHV using a virus microneutralization assay. The results demonstrate that TAHV transmission to mammals is widespread in Europe, particularly in the wild boar population where the mean rate of seroconversion is 15.2%.

The aim of this study was to determine the association of lung lesions with carcass and meat quality traits in slaughter pigs and to describe the main morphological features associated with lung lesions. Macroscopic lesions on the lungs were detected in 67.09% of a total of 79 pigs examined. Histopathological examination revealed that acute and chronic interstitial pneumonia represented the commonest changes, detected in 26.67% and 33.33% of the cases, respectively. Bronchopneumonia was found in 33.33% of the cases. By immunohistochemical examination, 26.67% of the lungs showed the presence of severe peribronchiolar and perialveolar infiltration composed predominantly of CD3+ T lymphocytes, which finding may be indicative of viral pneumonia. Regarding the production traits, it was confirmed that pigs with severe lung lesions had the lowest liveweight, hot carcass weight and meatiness, the highest pH value 45 min after slaughtering (pH45) and the highest incidence of dark, firm, dry (DFD) and pale, soft, exudative (PSE) meat. The presence of lung lesions significantly downgraded carcass value and caused a significant deterioration in pork quality.

Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded tissue samples of a cat that had suffered from disseminated adenovirus infection. The identity of the amplified products from the hexon and DNA-dependent DNA polymerase genes was confirmed by DNA sequencing. The sequences were clearly distinguishable from corresponding hexon and polymerase sequences of other mastadenoviruses, including human adenoviruses. These results suggest the possible existence of a distinct feline adenovirus.

Background Despite spectacular successes in hepatitis B and C therapies, severe hepatic impairment is still a major treatment problem. The clinically tested infectious bursal disease virus (IBDV) superinfection therapy promises an innovative, interferon-free solution to this great unmet need, provided that a consistent manufacturing process preventing mutations or reversions to virulent strains is obtained.Methods To address safety concerns, a tissue culture adapted IBDV vaccine strain V903/78 was cloned into cDNA plasmids ensuring reproducible production of a reverse engineered virus R903/78. The therapeutic drug candidate was characterized by immunocytochemistry assay, virus particle determination and immunoblot analysis. The biodistribution and potential immunogenicity of the IBDV agent was determined in mice, which is not a natural host of this virus, by quantitative detection of IBDV RNA by a quantitative reverse transcriptase-polymerase chain reaction and virus neutralization test, respectively.Results Several human cell lines supported IBDV propagation in the absence of visible cytopathic effect. The virus was stable from pH8 to pH6 and demonstrated significant resistance to low pH and also proved to be highly resistant to high temperatures. No pathological effects were observed in mice. Single and multiple oral administration of IBDV elicited antibodies with neutralizing activities in vitro.Conclusions Repeat oral administration of R903/78 was successful despite the presence of neutralizing antibodies. Single oral and intravenous administration indicated that IBDV does not replicate in mammalian liver alleviating some safety related concerns. These data supports the development of an orally delivered anti-hepatitis B virus/ anti-hepatitis C virus viral agent for human use. Copyright (c) 2015 John Wiley & Sons, Ltd.

Bovine viral diarrhoea (BVD) is a viral disease appearing in various forms and causing high economic losses in the cattle stocks of Hungary. The aim of the present study was to determine the prevalence of bovine viral diarrhoea virus (BVDV) in Hungary through a monitoring survey carried out on samples collected in cattle-keeping units throughout the country. Since no such survey had been carried out in Hungary during the last thirty years, our study may serve as a basis for later monitoring investigations aimed at following the progress of an expected eradication campaign of BVD. The tests were carried out using an ELISA method, on a total of 1200 blood samples submitted from 54 cattle herds. The herds had not been vaccinated against BVDV before the sampling. Out of the 1200 samples, 521 proved to be positive (43.4%), 40 gave doubtful result (3.3%) and 639 were negative (53.3%). In some stocks the samples were collected from cows having completed several lactation periods, and therefore the seronegativity indicates the BVDV-free status of the given stock. Moreover, among the positive herds we found a few where the seropositivity rate was rather low (< 5%). According to the results of the survey, a rather high portion (about one third) of the cattle-keeping units of Hungary can be regarded as BVDV free, which ratio is much higher than had been expected on the basis of surveys carried out on a lower number of samples and in smaller regions of the country. Hence, the chances of an eradication campaign launched in the near future, or carried out parallel to the IBR eradication programme, are better than previously expected.

We describe the detection and successful isolation of a novel mastadenovirus from a pygmy marmoset (Callithrix [Cebuella] pygmaea) that died following an episode of severe respiratory signs. Pathologic/histopathologic examination revealed hydrothorax and catarrhal bronchopneumonia with pronounced desquamation of the bronchiolar epithelial cells, while in other airways a marked hyperplasia of the epithelial lining and numerous giant cells could be observed. We obtained partial sequence data from the adenoviral DNA-dependent DNA-polymerase gene of the isolated strain and analyses of this region showed the highest level of identity to the recently described bat adenoviruses (strains PPV1 and TJM) and the type 2 canine adenovirus. Similar results were gained by phylogenetic calculations indicating that this novel marmoset adenovirus is only distantly related to reference Old and New World primate adenoviruses and formed a monophyletic group with bat and canine adenoviruses and the equine adenovirus 1. Even though the source of the infection remained unknown, our results could imply the possibility of a cross-species transmission of the virus from an anonymous host to the pygmy marmoset.

Viral hepatitis caused by Hepatitis E Virus (HEV), an enterically transmitted human pathogen is mainly associated with acute, self-limited, icteric hepatitis with an average of 1% mortality. Animal reservoirs are suspected to play role in the maintenance of the virus and in the spread of HEV to humans, having account not only in food-hygiene but also in workplace health and safety. Although, up to now clinical hepatitis in animals has not been observed, HEV-induced seroconversion was described in several species. HEV strains of animal and human origins are genetically closely related, which supports the suspicion on the zoonotic transmission of the virus.In the authors' survey done between 2005 and 2009 the occurrence of HEV was investigated in samples of animal origin collected in Hungary. HEV RNA was detected by reverse transcription-polymerase chain reaction in liver and faeces samples of domestic swine belonging to different age groups, wild boar, red deer and roe deer. 11% of samples of examined wild boars, 22% of samples of roe deer and 10% of samples of red deer were positive. Samples of domestic ruminants, as well as rodents and common shrew collected in swine farms, forests and meadows were tested negative for HEV RNA.Phylogenetic analysis of partial sequences of the viral nucleic acid of selected strains revealed that the investigated viruses belong to 3 subgroups of the third genogroup of HEV, and are closely related to HEV strains of human and animal origins detected in different countries. The investigations revealed widespread distribution of HEV in animals in Hungary, and considerable genetic diversity amongst the strains.