West Nile virus infections in Hungary: Epidemiological update and phylogenetic analysis of the Hungarian virus strains between 2015 and 2022.

Following the introduction of the West Nile virus (WNV) into Hungary in 2004, it has shortly become one of the most important human arbovirus infections, with a gradually increasing number of cases. The study aimed to summarize the current epidemiological situation in Hungary and sequence the WNV PCR-positive clinical specimens and virus isolates by next-generation whole genome sequencing (NGS) to obtain a detailed phylogenetic analysis of the circulating virus strains. Whole blood and urine samples from confirmed WNV-infected patients and WNV isolates were investigated by reverse transcription PCR assays. Genome sequencing was carried out by Sanger-method, followed by NGS on the Illumina MiSeq platform. Altogether 499 human infections were diagnosed between 2004 and 2022. A particularly remarkable increase in human WNV infections was observed in 2018, while the number of reported cases significantly decreased during the COVID-19 pandemic. Between 2015 and 2022, 15 WNV isolates, and 10 PCR-positive clinical specimens were investigated by NGS. Phylogenetic analysis revealed that the major European WNV lineage 2 clades, namely the Eastern European (or Russian) and the Central European (or Hungarian) clades, are presented in Hungary. Strains of the Balkan and other European clusters within the Central European clade are co-circulating in the country, following a characteristic geographical distribution. In Hungary, the presence and co-circulation of multiple lineage 2 WNV strains could be identified in the last few years. Therefore, in light of the 2018 WNV outbreak, sequence-based typing of the currently circulating strains could highly support outbreak investigations.

Extraordinary increase in West Nile virus cases and first confirmed human Usutu virus infection in Hungary, 2018.

BackgroundDuring the 2018 WNV transmission season, similarly to other endemic areas in Europe, a large number of human West Nile virus (WNV) infections were reported in Hungary.AimsWe summarise the epidemiological and laboratory findings of the 2018 transmission season and expand experiences in flavivirus differential diagnostics.MethodsEvery patient with clinical suspicion of acute WNV infection was in parallel tested for WNV, tick-borne encephalitis virus and Usutu virus (USUV) by serological methods. Sera, whole blood and urine samples were also tested for the presence of viral nucleic acid.ResultsUntil the end of December 2018, 215 locally acquired and 10 imported human WNV infections were notified in Hungary. All reported cases were symptomatic; most of them exhibited neurological symptoms. In a large proportion of tested individuals, whole blood was the most appropriate sample type for viral nucleic acid detection, but because whole blood samples were not always available, testing of urine samples also extended diagnostic possibilities. In addition, the first human USUV infection was confirmed in 2018 in a patient with aseptic meningitis. Serological cross-reactions with WNV in different serological assays were experienced, but subsequent molecular biological testing and sequence analysis identified Europe lineage 2 USUV infection.ConclusionCareful interpretation and simultaneous application of different laboratory methods are necessary to avoid misdiagnosis of human USUV cases. Expansion of the laboratory-confirmed case definition criteria for detection of viral RNA in any clinical specimens to include urine samples could increase diagnostic sensitivity.

Early start of the West Nile fever transmission season 2018 in Europe.

In Europe, surveillance indicates that the 2018 West Nile fever transmission season started earlier than in previous years and with a steeper increase of locally-acquired human infections. Between 2014 and 2017, European Union/European Economic Area (EU/EEA) and EU enlargement countries notified five to 25 cases in weeks 25 to 31 compared with 168 cases in 2018. Clinicians and public health authorities should be alerted to ensure timely implementation of prevention measures including blood safety measures.

Lymphocytic Choriomeningitis Virus Infections in Hungary between 2017-2023-Investigation of the First Congenital Infections.

Lymphocytic choriomeningitis virus (LCMV) is a neglected rodent-borne arenavirus, primarily spread by common house mouse species. Acquired human infections range from asymptomatic to mild flu-like symptoms and self-resolving neurological diseases. In contrast, intrauterine LCMV infection is associated with high mortality and morbidity. Infection of the fetus often leads to fetal death, and surviving fetuses may develop vision impairment and central nervous system developmental disorders. LCMV is mainly diagnosed by serological methods using in-house indirect immunofluorescence assays. LCMV nucleic acid is detected by the nested RT-PCR method and confirmed by Sanger sequencing. In Hungary, 23 acquired lymphocytic choriomeningitis cases were diagnosed between 2017 and 2023. Ten out of 23 confirmed patients proved to be positive by the PCR method. Two cases of intrauterine LCMV infections were detected in 2019 and 2021, respectively. The IgG antibody titers measured in the infant's serum samples were much higher than the IgG titers of the maternal serum samples. Both IgM and IgA antibodies were detectable in the infants' sera. As the microbiological diagnosis of LCMV is rather challenging and the symptoms are very similar to the clinical picture of other common teratogenic pathogens such as cytomegalovirus or Toxoplasma gondii, intrauterine LCMV infections might still be underdiagnosed.

West Nile and Usutu virus seroprevalence in Hungary: A nationwide serosurvey among blood donors in 2019.

In Hungary, West Nile virus (WNV) has been responsible for 459 laboratory confirmed human cases between 2004 and 2019, while the first human Usutu virus (USUV) infection was confirmed only in 2018. A comprehensive serosurvey was conducted among blood donors to assess the WNV and USUV seroprevalence in 2019, one year after the largest European WNV epidemic. Altogether, 3005 plasma samples were collected and screened for WNV and USUV specific Immunoglobulin G (IgG) antibodies by Enzyme-Linked Immunosorbent Assay (ELISA). All reactive samples were further tested for tick-borne encephalitis virus IgG antibodies by ELISA. Indirect immunofluorescence test and microneutralization assay were used as confirmatory methods. Overall, the WNV seroprevalence was 4.32%, and in five blood donors USUV seropositivity was confirmed. The highest seroprevalence was measured in Central, Eastern and Southern Hungary, while the Western part of the country proved to be less affected. There was a statistically strong association between the WNV seroprevalence of 2019 and the cumulative incidence in the period of 2004 and 2019 calculated for every NUTS 3 region. The last WNV serological screening was performed in 2016 and the prevalence of anti-WNV IgG proved to be 2.19%. One year after the 2018 WNV outbreak, a significant increase in seroprevalence was observed in the Hungarian population and evidence for USUV seropositivity was also obtained. The spatial pattern of seroprevalence can support the identification of high-risk areas raising awareness of the need for increased surveillance, such as screening vector, equine, and avian populations. The communication with general practitioners and other professionals in primary health care services can support the early identification of acute human cases. Education and awareness-raising on the importance of protection against mosquito vectors amongst residents are also important parts of preventive measures.

Multi-Approach Investigation Regarding the West Nile Virus Situation in Hungary, 2018.

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.