Viktória Németh

Molecular survey of RNA viruses in Hungarian bats: discovering novel astroviruses, coronaviruses, and caliciviruses.

UNLABELLED Background: Bat-borne viruses pose a potential risk to human health and are the focus of increasing scientific interest. To start gaining information about bat-transmitted viruses in Hungary, we tested multiple bat species for several virus groups between 2012 and 2013. MATERIALS AND METHODS Fecal samples were collected from bats across Hungary. We performed group-specific RT-PCR screening for astro-, calici-, corona-, lyssa-, othoreo-, paramyxo-, and rotaviruses. Positive samples were selected and sequenced for further phylogenetic analyses. RESULTS A total of 447 fecal samples, representing 24 European bat species were tested. Novel strains of astroviruses, coronaviruses, and caliciviruses were detected and analyzed phylogenetically. Out of the 447 tested samples, 40 (9%) bats were positive for at least one virus. Bat-transmitted astroviruses (BtAstV) were detected in eight species with a 6.93% detection rate (95% confidence interval [CI] 4.854, 9.571). Coronaviruses (BtCoV) were detected in seven bat species with a detection rate of 1.79% (95% CI 0.849, 3.348), whereas novel caliciviruses (BtCalV) were detected in three bat species with a detection rate of 0.67% (95% CI 0.189, 1.780). Phylogenetic analyses revealed a great diversity among astrovirus strains, whereas the Hungarian BtCoV strains clustered together with both alpha- and betacoronavirus strains from other European countries. One of the most intriguing findings of our investigation is the discovery of novel BtCalVs in Europe. The Hungarian BtCalV did not cluster with any of the calcivirus genera identified in the family so far. CONCLUSIONS We have successfully confirmed BtCoVs in numerous bat species. Furthermore, we have described new bat species harboring BtAstVs in Europe and found new species of CalVs. Further long-term investigations involving more species are needed in the Central European region for a better understanding on the host specificity, seasonality, phylogenetic relationships, and the possible zoonotic potential of these newly described viruses.

West Nile virus surveillance in mosquitoes, April to October 2013, Vojvodina province, Serbia: implications for the 2014 season.

After the West Nile virus (WNV) outbreak in 2012, we collected mosquito samples from Vojvodina province, Serbia, in 2013. We found high WNV infection rate in two species, Culex pipiens and Anopheles maculipennis. Phylogenetic analysis showed that Serbian WNV strains from 2013 were most closely related to Italian and Greek strains isolated in 2012 and 2010, respectively. Public health authorities should be aware of a potentially increased risk of WNV activity during the 2014 season.

Detection of Dobrava-Belgrade hantavirus using recombinant-nucleocapsid-based enzyme-linked immunosorbent assay and SYBR Green-based real-time reverse transcriptase-polymerase chain reaction

Dobrava (DOBV) hantaviruses belong to the genus Hantavirus, family Bunyaviridae, and are carried by yellow-necked and striped field mice. The goal of this study was to detect DOBV using serological and genetic methods in Apodemus rodents in Hungary and in northern Croatia. During the study period, a total of 125 Apodemus sp. (67 A. agrarius, 58 A. flavicollis) were tested for the presence of hantaviruses, and 21 rodents (17%) were positive by rRT-PCR and/or ELISA. We conclude that the prevalence of DOBV is much higher than previously anticipated. The simultaneous use of molecular and serological techniques provides a highly reliable way to detect hantavirus infections.

Serologic Evidence of Crimean-Congo Hemorrhagic Fever Virus Infection in Hungary

Crimean-Congo hemorrhagic fever virus (CCHFV) is a typical tick-borne pathogen that causes an increasing number of severe infections in many parts of Africa, Asia, the Middle East, and the Balkans, as well as in some other parts of Europe. The virus is transmitted primarily by Hyalomma spp., and the spectrum of natural hosts for CCHFV is broad, including wild and domestic animals. Although, the presence of CCHFV was hypothesized in Hungary, no significant research activity has been carried out in the past 30 years. In the present study, we provide serological evidence of CCHFV infection in Lepus europeus using newly developed antibody detection assays. Of 198 samples, 12 (6%) were positive for immunoglobulin G antibody against CCHFV, with 2 independent detection assays. This observation indicates a need for a large-scale surveillance to estimate the potential public health risk of CCHFV in Hungary.

Identification of tick-borne encephalitis virus in ticks collected in southeastern Hungary

Tick-borne encephalitis virus (TBEV) is an arthropod-borne viral pathogen causing infections in Europe and is responsible for most arbovirus central nervous system infections in Hungary. Assessing the TBEV prevalence in ticks through detection of genomic RNA is a broadly accepted approach to estimate the transmission risk from a tick bite. For this purpose, 2731 ticks were collected from the neighboring area of the town of Dévaványa, located in southeastern Hungary, which is considered a low-risk-transmission area for TBEV. Altogether, 2300 ticks were collected from the vegetation, while 431 were collected from rodents. Samples were pooled and then screened for TBEV with a newly designed semi-nested RT-PCR (RT-snPCR) targeting the NS1 genomic region. PCR results were confirmed by direct sequencing of the second round amplicons. Among the 3 different collected tick species (Ixodes ricinus, Haemaphysalis concinna, Dermacentor marginatus), I. ricinus was the only species that tested positive for TBEV. TBEV-positive ticks were collected from small mammals or from the vegetation. One nymphal pool and 4 larval pools tested positive for TBEV. The only positive nymphal pool was unfed and came from vegetation, while ticks of the 4 positive larval pools were collected from rodents. Minimal TBEV prevalence in ticks was 0.08% for unfed nymphs and 0.78% for feeding larvae. Our results indicate that further long-term investigations on the occurrence of TBEV are needed to better describe the geographic distribution and the prevalence of infected ticks in Hungary.

Putative novel lineage of West Nile virus in Uranotaenia unguiculata mosquito, Hungary.

West Nile virus (WNV) is an increasing public health concern in Europe with numerous human cases. A total of 23,029 female mosquitoes were tested for a variety of mosquito-borne flaviviruses and orthobunyaviruses supposedly endemic in Southern Transdanubia, Hungary, in the frames of a large-scale surveillance between 2011 and 2013. WNV nucleic acid was detected in a single pool containing Uranotaenia unguiculata mosquitoes. Sequence- and phylogenetic analyses for two different regions (NS5 and E) of the viral genome showed that the novel Hungarian WNV strain was different from other previously described WNV lineages. These findings may indicate the presence of a putative, novel lineage of WNV in Europe. Our results also indicate that U. unguiculata mosquito may become relevant species as a potential vector for West Nile virus in Europe.

Dobrava–Belgrade hantavirus infection mimics acute appendicitis

Dobrava-Belgrade hantavirus infection mimicked acute appendicitis in a patient suffering from hemorrhagic fever with renal syndrome in Hungary. The 27-year-old man was admitted to the local hospital with severe abdominal pain localized mainly at the right lower quadrant of the abdomen and with fever, nausea, vomiting and bloody diarrhea. Based on these findings supported by computerized tomography acute perforated appendicitis was suspected and an explorative laparatomy was performed, which did not confirm the diagnosis. Next day he developed acute oliguric renal failure raising the possibility of hantavirus infection. Specific serum IgG and IgM antibodies against hantavirus were identified, and by molecular methods the presence of Dobrava-Belgrade virus was proven. This report describes a rare clinical manifestation of hemorrhagic fever with renal syndrome (HFRS), and shows that HFRS might be difficult to diagnose especially when symptoms mimick those of an acute abdominal inflammation.

Serologic survey of orthopoxvirus infection among rodents in Hungary

As a result of discontinuing vaccination against smallpox after the late 1970s, different orthopoxviruses (OPVs), such as cowpox virus (CPXV), have become a re-emerging healthcare threat among zoonotic pathogens. In Hungary, data on OPV prevalence among its rodent host species have been absent. Here, rodents belonging to four species, i.e., striped field mouse (Apodemus agrarius), yellow-necked mouse (A. flavicollis), wood mouse (A. sylvaticus) and bank vole (Myodes glareolus), were live trapped at 13 sampling plots on a 149-ha area in the Mecsek Mountains, Hungary, from March to September in 2011 and 2012. Rodent sera were collected and screened for OPV-reactive antibodies with an immunfluorescence assay (IFA). Among the 1587 tested rodents, 286 (18.0%) harbored OPV-specific antibodies. Seroprevalence was the highest for the bank vole (71.4%) and the striped field mouse (66.7%). Due to a masting event in the autumn of 2011 across Central Europe, the abundance of bank voles increased drastically in the 2012 season, raising the overall OPV seroprevalence. We provide the first data on OPV occurrence and seroprevalence in rodents in Hungary. The circulation of OPV in rodents in densely populated areas warrants further studies to elucidate the zoonotic potential of OPV in humans.

Molecular characterization of Dobrava and Kurkino genotypes of Dobrava-Belgrade hantavirus detected in Hungary and Northern Croatia

Among the Hantavirus genus, Saaremaa virus (SAAV) has been the subject of taxonomical debates. While the International Committee on Taxonomy of Viruses declares SAAV as a distinct species, several European hantavirus experts proposed that SAAV is in fact a genotype of Dobrava-Belgrade virus (DOBV). In the present study we performed S-segment-based phylogenetic analysis of eight DOBV strains identified in rodents in Hungary and Northern Croatia. These new sequences considerably increase the number of complete nucleoprotein gene sequences deposited in the NCBI database. Our phylogenetic analysis clearly support the taxonomical nomenclature recently proposed for DOBV, i.e., genotypes such as Dobrava, Saaremaa, Kurkino, and Sochi should indeed be classified within the DOBV hantavirus species. Moreover, we found that only the Dobrava and Kurkino genotypes of DOBV species are circulating in Hungary while currently there is no evidence for the presence of Saaremaa genotype.

Serosurvey of pathogenic hantaviruses among forestry workers in Hungary.

ObjectivesThe aim of the study was to survey the prevalence of human hantavirus infections among forestry workers, who are considered a risk population for contracting the disease. Sera collected from volunteers were tested for antibodies against Dobrava-Belgrade (DOBV) and Puumala (PUUV) viruses.Material and MethodsFor serological analyses, full capsid proteins of DOBV and PUUV viruses were produced in a bacterial expression system, while Ni-resin was used for protein purification. Samples were screened for anti-hantavirus antibodies by ELISA, results were confirmed by Western blot analysis.ResultsA total of 835 samples collected from 750 males and 85 females were tested by indirect ELISA and positive test results were confirmed by Western blot assay. Out of the 45 ELISA-reactive samples, 38 were confirmed by Western blot analysis. The regional distribution of seropositive individuals was as follows: 1.9% (2/107) in the Danube-Tisza Plateau (Great Plains), 3.1% (10/321) in the Southern Transdanubian region, 5.2% (13/248) in the Northern Transdanubian, and 8.2% (13/159) in the North Hungarian Mountains.ConclusionsOur data show marked geographic differences in seroprevalence of pathogenic hantaviruses within Hungary, indicating elevated exposure to hantavirus infections in some areas.