Sequencing and molecular modeling identifies candidate members of Caliciviridae family in bats.

Emerging viral diseases represent an ongoing challenge for globalized world and bats constitute an immense, partially explored, reservoir of potentially zoonotic viruses. Caliciviruses are important human and animal pathogens and, as observed for human noroviruses, they may impact on human health on a global scale. By screening fecal samples of bats in Hungary, calicivirus RNA was identified in the samples of Myotis daubentonii and Eptesicus serotinus bats. In order to characterize more in detail the bat caliciviruses, large portions of the genome sequence of the viruses were determined. Phylogenetic analyses and molecular modeling identified firmly the two viruses as candidate members within the Caliciviridae family, with one calicivirus strain resembling members of the Sapovirus genus and the other bat calicivirus being more related to porcine caliciviruses of the proposed genus Valovirus. This data serves the effort for detecting reservoir hosts for potential emerging viruses and recognize important evolutionary relationships.

Circulation of Dirofilaria repens, Setaria tundra, and Onchocercidae species in Hungary during the period 2011-2013.

Dirofilaria repens and recently Dirofilaria immitis are known to be endemic in Hungary. Since there is no related research on Dirofilaria carrier mosquito species from Hungary, we conducted a three-year mosquito surveillance study between 2011 and 2013. During the study period we examined 23,139 female mosquitoes with a generic filaria-specific TaqMan PCR assay, and characterized them by sequencing a 500 bp segment of 12S rRNA. An important result of our study was the detection of Setaria tundra and D. repens along with an unidentified Onchocercidae nematode. D. repens is known to be endemic in Hungary, however, the detection of S. tundra in all sampling sites throughout the study period indicates for the first time the endemicity of this parasite in Hungary. The Onchocercidae sp. nematode showed 95% nucleotide identity with previously detected unidentified nematodes from Germany, indicating a broader geographical distribution of this nematode in Europe. D. immitis specific DNA was not detected among the screened mosquitoes in this study. Here we report 11 mosquito species as potential vector organisms for local filarial infections, including Aedes vexans, Ochlerotatus annulipes, Ochlerotatus sticticus, Coquillettidia richiardii, Anopheles hyrcanus and Ochlerotatus rusticus. Dirofilaria development unit was calculated and the potential transmission period was estimated, which ranged between 65 and 113 days between sampling seasons. A relatively high infection rate (36.8%) was identified, which is a notable finding for veterinary and human health professionals. Moreover, the results of our study widen the group of possible mosquito vector species for D. repens and S. tundra in Central Europe.

Genetic diversity and recombination within bufaviruses: Detection of a novel strain in Hungarian bats.

Bats are important hosts of many viruses and in several cases they may serve as natural reservoirs even for viruses with zoonotic potential worldwide, including Europe. However, they also serve as natural reservoir for other virus groups with important evolutionary relevance and yet unknown zoonotic potential. We performed viral metagenomic analyses on Miniopterus schreibersii bat fecal samples. As a result, a novel parvovirus was detected for the first time in European bats. Although, bufavirus was recently discovered as a novel human infecting parvovirus, here we report sequence data of the first bufavirus from European bats related to human bufaviruses. Based on our sequence data a possible intragenic recombination event was detected within bufaviruses which may serves as an important milestone in their evolution.

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.

Genetic characterization of a novel picornavirus detected in Miniopterus schreibersii bats.

Bats are important reservoirs of many viruses with zoonotic potential worldwide, including Europe. Among bat viruses, members of the Picornaviridae family remain a neglected group. We performed viral metagenomic analyses on Miniopterus schreibersii bat faecal samples, collected in Hungary in 2013. In the present study we report the first molecular data and genomic characterization of a novel picornavirus from the bat species M. schreibersii in Europe. Based on phylogenetic analyses, the novel bat picornaviruses unambiguously belong to the Mischivirus genus and were highly divergent from other bat-derived picornaviruses of the Sapelovirus genus. Although the Hungarian viruses were most closely related to Mischivirus A, they formed a separate monophyletic branch within the genus.

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.

Serosurvey of pathogenic hantaviruses among forestry workers in Hungary.

OBJECTIVES: The 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 METHODS: For 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. RESULTS: A 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. CONCLUSIONS: Our data show marked geographic differences in seroprevalence of pathogenic hantaviruses within Hungary, indicating elevated exposure to hantavirus infections in some areas.

Molecular detection and phylogenetic analysis of tick-borne encephalitis virus in rodents captured in the transdanubian region of Hungary.

Abstract Tick-borne encephalitis virus (TBEV) infection is a common zoonotic disease affecting humans in Europe and Asia. To determine whether TBEV is present in small mammalian hosts in Hungary, liver samples of wild rodents were tested for TBEV RNA. Over a period of 7 years, a total of 405 rodents were collected at five different geographic locations of the Transdanubian region. TBEV nucleic acid was identified in four rodent species: Apodemus agrarius, A. flavicollis, Microtus arvalis, and Myodes glareolus. Out of the 405 collected rodents, 17 small mammals (4.2%) were positive for TBEV. The present study provides molecular evidence and sequence data of TBEV from rodents in Hungary.

Identification of hantavirus infection by Western blot assay and TaqMan PCR in patients hospitalized with acute kidney injury.

Hantaviruses, one of the causative agents of viral hemorrhagic fevers, represent a considerable healthcare threat. In Hungary, Dobrava-Belgrade virus (DOBV) and Puumala virus (PUUV) are the main circulating hantavirus species, responsible for the clinical picture known as hemorrhagic fever with renal syndrome, a disease that may be accompanied by acute kidney injury (AKI), requiring hospitalization with occasionally prolonged recovery phase. A total of 20 patient sera were collected over a 2-year period from persons hospitalized with AKI, displaying clinical signs and laboratory findings directly suggestive for hantavirus infection. Samples were tested using an immunoblot assay, based on complete viral nucleocapsid proteins to detect patients' IgM and IgG antibodies against DOBV and PUUV. In parallel, all specimens were also tested by 1-step real-time TaqMan reverse-transcriptase polymerase chain reaction to confirm infection and to determine the causative hantavirus genotype. We present here the first Hungarian clinical study spanning across 2 years and dedicated specifically to assess acute kidney injuries, in the context of hantavirus prevalence.

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.

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.

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.