Isolation and complete genome characterization of novel reassortant orthoreovirus from common vole (Microtus arvalis).

A novel mammalian orthoreovirus (MRV) strain was isolated from the lung tissue of a common vole (Microtus arvalis) with Tula hantavirus infection. Seven segments (L1-L3, M2-M3, S2, and S4) of the Hungarian MRV isolate MORV/47Ma/06 revealed a high similarity with an MRV strain detected in bank vole (Myodes glareolus) in Germany. The M1 and S3 segment of the Hungarian isolate showed the closest relationship with the sequence of a Slovenian human and a French murine isolate, respectively. The highest nucleotide and amino acid identity values were above 90 and 95% in all of the comparisons to the reference sequences in GenBank, except for the S1 with a maximum of 69.6% nucleotide and 75.4% amino acid identity. As wild rodents are among the main sources of zoonotic infections, the reservoir role of these animals and zoonotic potential of rodent origin MRVs need to be further investigated.

Phylogenetic evidence for a new species of Barbus in the Danube River basin.

Three species of small-sized rheophilic Barbus fishes are endemic to and widely distributed throughout the mountain regions in the Danube River basin. In Hungary, barbels referred to as B. petenyi occur in streams in the foothills of the Carpathians near the borders with Slovakia, Ukraine and Romania. However, up to now, no genetic investigations were carried out on rheophilic barbels in this region. This study aims to clarify the taxonomic identity and distribution of the rheophilic barbels in the Hungarian plain based on molecular and morphological analyses. Two mitochondrial genes (cytochrome b, ATPase 6/8) and one nuclear gene (beta-actin intron 2) were sequenced and several morphometric and meristic characters were recorded. Phylogenetic and morphological analyses revealed that there are four genetically distinct lineages among the rheophilic barbels in the Carpathian Basin. The results demonstrated that North-Hungarian Barbus populations belong to B. carpathicus and that B. petenyi presumably does not occur in Hungary. As expected, B. balcanicus was only recorded in samples from the Balkans analyzed for reference. A distinct species, new to science, was discovered to be present in Sebes-Körös River (Crisul Repede) in eastern Hungary and western Romania and is formally described here as B. biharicus Antal, László, Kotlík - sp. nov.

First molecular identification of Dirofilaria spp. (Onchocercidae) in mosquitoes from Serbia.

Dirofilariosis is a common and widespread veterinary health issue in several European countries with notable zoonotic potential. The causative agents are Dirofilaria immitis and Dirofilaria repens nematoda species which are transmitted by different mosquito vectors. Similar to other mosquito-borne infections, the knowledge about mosquito species involved in disease transmission is crucial for the complex understanding of local transmission cycles. Since there is no available data on mosquito species, potentially involved in disease transmission from Serbia, 6369 female mosquito individuals were retrospectively tested for Dirofilaria nematodes, collected from 13 localities in Vojvodina province, Serbia, in 2013. Altogether, 8.33 % of tested pools showed positivity, composed of five mosquito species, mainly, Culex pipiens and Aedes vexans. D. immitis and D. repens were both detected from multiple localities, during the whole period of mosquito breeding season, which provides the first data on local transmission characteristics regarding mosquitoes from the Balkans.

Candidate new rotavirus species in sheltered dogs, Hungary.

We identified unusual rotavirus strains in fecal specimens from sheltered dogs in Hungary by viral metagenomics. The novel rotavirus species displayed limited genome sequence homology to representatives of the 8 rotavirus species, A-H, and qualifies as a candidate new rotavirus species that we tentatively named Rotavirus I.

Canine rotavirus C strain detected in Hungary shows marked genotype diversity.

Species C rotaviruses (RVC) have been identified in humans and animals, including pigs, cows and ferrets. In dogs, RVC strains have been reported anecdotally on the basis of visualization of rotavirus-like virions by electron microscopy combined with specific electrophoretic migration patterns of the genomic RNA segments. However, no further molecular characterization of these viruses was performed. Here, we report the detection of a canine RVC in the stool of a dog with enteritis. Analysis of the complete viral genome uncovered distinctive genetic features of the identified RVC strain. The genes encoding VP7, VP4 and VP6 were distantly related to those of other RVC strains and were putatively classified as G10, P8 and I8, respectively. The new strain was named RVC/Dog-wt/HUN/KE174/2012/G10P[8]. Phylogenetic analyses revealed that canine RVC was most closely related to bovine RVC strains with the exception of the NSP4 gene, which clustered together with porcine RVC strains. These findings provide further evidence for the genetic diversity of RVC strains.

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.

Complete genome analysis identifies Tvärminne avian virus as a candidate new species within the genus Orthoreovirus.

Orthoreoviruses have been associated with a variety of diseases in domesticated poultry and wild-living birds. In 2002, a reovirus strain named Tvärminne avian virus (TVAV), was identified in Finland in a crow showing neurological disorders. The objective of this study was the molecular characterization of this novel reovirus strain. Genome sequencing was performed by combining semiconductor sequencing and traditional capillary sequencing. Sequence and phylogenetic analyses showed that TVAV shares low nucleotide sequence identity with other reoviruses (range for each gene, 31-72 %) including strains belonging to the species Avian orthoreovirus. The most closely related reovirus strain was an isolate identified in Steller sea lion. Our data indicate that TVAV is a divergent reovirus of avian origin that may be the first representative of a distinct virus species within the genus Orthoreovirus.

The complete genome sequence of a European goose reovirus strain.

The complete genomic sequence of a Hungarian goose orthoreovirus strain (D20/99) is reported in this study. The genome of D20/99 is 22,969 bp in length (range, 3958 bp for L1 to 1124 bp for S4) and encodes 11 putative proteins. Pairwise sequence comparisons and phylogenetic analyses indicated that D20/99 shares genetic signatures with some contemporary Chinese duck and goose reovirus strains, except for the µA, µNS and σA protein coding genes, which represented independent genetic lineages. This study implies a greater genetic diversity among waterfowl-origin orthoreoviruses than hitherto recognized.

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.

Analysis and Comparison of Rapid Methods for the Determination of Ochratoxin a Levels in Organs and Body Fluids Obtained from Exposed Mice.

Mycotoxins are bioaccumulative contaminants impacting animals and humans. The simultaneous detection of frequent active exposures and accumulated mycotoxin level (s) in exposed organisms would be the most ideal to enable appropriate actions. However, few methods are available for the purpose, and there is a demand for dedicated, sensitive, reliable, and practical assays. To demonstrate the issue, mice were exposed to a relevant agent Ochratoxin A (OTA), and accumulated OTA was measured by fine-tuned commercial assays. Quantitative high-performance liquid chromatography with fluorescence detection, enzyme-linked immunosorbent assay, and flow cytometry assays have been developed/modified using reagents available as commercial products when appropriate. Assays were performed on excised samples, and results were compared. Accumulated OTA could be detected and quantified; positive correlations (between applied doses of exposure and accumulated OTA levels and the results from assays) were found. Dedicated assays could be developed, which provided comparable results. The presence and accumulation of OTA following even a short exposure could be quantitatively detected. The assays performed similarly, but HPLC had the greatest sensitivity. Blood contained higher levels of OTA than liver and kidney. We demonstrate that specific but flexible and practical assays should be used for specific/local purposes, to measure the exposure itself and accumulation in blood or organs.

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.

Temporal Dynamics of Two Pathogenic Hantaviruses Among Rodents in Hungary.

Hantaviruses are worldwide pathogens, which often cause serious or even fatal diseases in humans. Hosts are predominantly in the form of rodents and soricomorphs; however, bats are also described as an important reservoir. In Hungary, representatives of two human pathogenic species of the genus Orthohantavirus are present: the Dobrava-Belgrade orthohantavirus and Puumala orthohantavirus. In Hungarian forests, the dominant rodent species are Apodemus flavicollis, Apodemus agrarius, Apodemus sylvaticus, and Myodes glareolus, all of which are natural reservoirs comprising different hantaviruses. The aim of the study was to survey the prevalence of hantaviruses among rodent populations and examine the potential relationship regarding population densities, years, sex, and seroprevalence. Rodents were trapped at 13 sampling plots in a forest reserve located in the Mecsek Mountain range, Hungary, from March to October between 2011 and 2014. Rodent serum samples were tested for IgG antibodies against Dobrava-Belgrade virus and Puumala virus by enzyme-linked immunosorbent assay (ELISA) using a recombinant nucleocapsid protein. During the 4-year sampling period, 2491 specimens were tested and 254 (10.2%) proved seropositive for orthohantaviruses. In 2011, the seroprevalence among Apodemus spp. and M. glareolus was 17.2% (114/661) and 3.9% (3/77), respectively, although this rate had reversed itself in 2014. Seropositivity was substantiated in 18.4% (12/65) of Myodes voles, while only 3.6% (13/359) of the tested Apodemus rodents were found to be IgG positive. Seroconversion was observed in 58 cases, while seroreversion was only detected in 3 individual cases. A significant difference among the number of infected males and females was identified in the first 2 years of our study. Winter survival with respect to rodents was not negatively affected due to the hantavirus infection. Hantavirus seroprevalence was not directly influenced by host abundance. Consequently, we assume that high rodent density alone does not lead to an increased risk of hantavirus infection among the rodent host population.

Candidate new rotavirus species in Schreiber's bats, Serbia.

The genus Rotavirus comprises eight species designated A to H and one tentative species, Rotavirus I. In a virus metagenomic analysis of Schreiber's bats sampled in Serbia in 2014 we obtained sequences likely representing novel rotavirus species. Whole genome sequencing and phylogenetic analysis classified the representative strain into a tentative tenth rotavirus species, we provisionally called Rotavirus J. The novel virus shared a maximum of 50% amino acid sequence identity within the VP6 gene to currently known members of the genus. This study extends our understanding of the genetic diversity of rotaviruses in bats.

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.

Genetic Characterization of Providence Virus Isolated from Bat Guano in Hungary.

We report the complete genome sequence and genetic characterization of a novel strain of Providence virus, detected in Barbastella barbastellus bat guano, collected in Hungary in 2014. Our data may facilitate the understanding of the evolutionary processes of this unique viral family of Carmotetraviridae.

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 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.

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.

Detection of shared genes among Asian and European waterfowl reoviruses in the whole genome constellations.

In order to explore the genetic relatedness and evolution of 'classical' and 'novel' waterfowl origin reoviruses (WRV) isolated in different years and continents, and filling up our lacking knowledge about the European WRV strains, the complete genomic sequence of two French isolates causing the 'classical' type of reovirus infection of Muscovy ducks had been determined. Based on the genome organization and the encoded proteins the two isolates could be referred as classical type strains. Sequence comparison showed that the two strains were most closely related to each other and belong to the same monophyletic group of European and Asian WRV strains. Phylogeny of the appropriate segments revealed potential reassortment events between waterfowl and chicken origin, and 'classical' and 'novel' and European and Chinese WRV strains. Our results point out a complex way of viral evolution regarding the origin and biological properties of the WRVs.