Metagenomic Identification of Novel Eukaryotic Viruses with Small DNA Genomes in Pheasants.

A panel of intestinal samples collected from common pheasants (Phasianus colchicus) between 2008 and 2017 was used for metagenomic investigation using an unbiased enrichment protocol and different bioinformatic pipelines. The number of sequence reads in the metagenomic analysis ranged from 1,419,265 to 17,507,704 with a viral sequence read rate ranging from 0.01% to 59%. When considering the sequence reads of eukaryotic viruses, RNA and DNA viruses were identified in the samples, including but not limited to coronaviruses, reoviruses, parvoviruses, and CRESS DNA viruses (i.e., circular Rep-encoding single-stranded DNA viruses). Partial or nearly complete genome sequences were reconstructed of at least three different parvoviruses (dependoparvovirus, aveparvovirus and chaphamaparvovirus), as well as gyroviruses and diverse CRESS DNA viruses. Generating information of virus diversity will serve as a basis for developing specific diagnostic tools and for structured epidemiological investigations, useful to assess the impact of these novel viruses on animal health.

Genetic characterisation of a novel reptarenavirus detected in a dead pet red-tailed boa (Boa constrictor).

Boid inclusion body disease (BIBD) is a severe and transmissible disease of snakes worldwide. Reptarenaviruses have been identified as the aetiological agents of BIBD. We determined the almost complete genome sequence of an arenavirus detected in a female red-tailed boa that had succumbed in a private collection in Hungary. We used a combination of next generation sequencing and Sanger sequencing methods. Based on the analysis of the obtained sequence data, the virus, tentatively named Coldvalley virus, seemed to belong to the Reptarenavirus genus of the Arenaviridae family. This classification was confirmed by the genome structure (bisegmented single-stranded RNA) characteristic of the genera Mammarenavirus and Reptarenavirus. The pairwise comparison of the nucleotide and amino acid sequences, as well as the topology of the maximum likelihood phylogenetic trees, suggested that the newly-characterised Coldvalley virus can be classified into the species Rotterdam reptarenavirus.

A novel gyrovirus in a common pheasant (Phasianus colchicus) with poult enteritis and mortality syndrome.

A novel gyrovirus was detected in an intestinal specimen of a common pheasant that died due to poult enteritis and mortality syndrome. The genome of the pheasant-associated gyrovirus (PAGyV) is 2353 nucleotides (nt) long and contains putative genes for the VP1, VP2, and VP3 proteins in an arrangement that is typical for gyroviruses. Gyrovirus-specific motifs were identified in both the coding region and the intergenic region of the PAGyV genome. The VP1 of PAGyV shares up to 67.6% pairwise nt sequence identity with reference sequences and forms a distinct branch in the phylogenetic tree. Thus, according to the recently described species demarcation criteria, PAGyV belongs to a novel species in the genus Gyrovirus, family Anelloviridae, for which we propose the name "Gyrovirus phaco 1".

Genomic characterization of avian and neoavian orthoreoviruses detected in pheasants.

Avian reoviruses are well-known pathogens seriously affecting the productivity of poultry industry. Game birds represent a small segment of the agricultural sector and much remained to be learnt about factors affecting productivity. Here we show that reovirus infections might occur in pheasants and demonstrate that reoviruses of pheasants are of diverse origin. The complete or coding-complete genomic sequences of two Hungarian reovirus strains, D1996/2/1 and Reo/HUN/Pheasant/216/2015, have been determined in this study. The strain D1996/2/1 was isolated in 2012 from birds with gizzard erosion, whereas the other strain was isolated in 2015 from diarrheic pheasant poults. Phylogenetic analyses showed that none of the Hungarian isolates shared common origin with a pheasant reovirus detected recently in the United States. Additionally, we found that Reo/HUN/Pheasant/216/2015 is a multi-reassortant reovirus within the species Avian orthoreovirus that shared genetic relationship with turkey reoviruses (σC), partridge reoviruses (λA, σB), and chicken reoviruses (λB, λC, µA, σA, and σNS), in the respective gene phylogenies, whereas two genes (µB and µNS) did not reveal any possible common ancestors. The other isolate, D1996/2/1, was found to be distantly related to previously described reoviruses raising the possibility that it might represent a novel orthoreovirus species or a new genogroup within the newly accepted species, Neoavian orthoreovirus. The genetic diversity among pheasant reoviruses could raise challenges for virus classification as well as for development of molecular diagnostic tools and vaccine based prevention and control measures.

Genomic sequence and phylogenetic analyses of two novel orthoreovirus strains isolated from Pekin ducks in 2014 in Germany.

Complete genomic sequences of two orthoreovirus strains, D2533/4/1-10 and D2533/6/1-10, isolated from Pekin ducklings in Germany have been determined. Pairwise sequence comparisons and phylogenetic analyses indicated that strain D2533/4/1-10 might have acquired its genomic segments from three different origins, from classical and novel waterfowl reoviruses, and a yet unknown orthoreovirus strain. D2533/6/1-10 proved to be only distantly related to previously described orthoreoviruses. Reassortment, host species transmission events, and successful adaptation of novel variants may signify a challenge for animal health and maintenance of economic production.

Novel adenovirus detected in kowari (Dasyuroides byrnei) with pneumonia.

A male kowari (Dasyuroides byrnei) originating from a zoo facility was delivered for post mortem evaluation in Hungary. Acute lobar pneumonia with histopathologic changes resembling an adenovirus (AdV) infection was detected by light microscopic examination. The presence of an AdV was confirmed by obtaining partial sequence data from the adenoviral DNA-dependent DNA-polymerase. Although the exact taxonomic position of this novel marsupial origin virus could not be determined, pairwise identity analyses and phylogenetic calculations revealed that it is distantly related to other members in the family Adenoviridae.

Whole genome sequencing of a rare rotavirus from archived stool sample demonstrates independent zoonotic origin of human G8P[14] strains in Hungary.

Genotype P[14] rotaviruses in humans are thought to be zoonotic strains originating from bovine or ovine host species. Over the past 30 years only few genotype P[14] strains were identified in Hungary totaling<0.1% of all human rotaviruses whose genotype had been determined. In this study we report the genome sequence and phylogenetic analysis of a human genotype G8P[14] strain, RVA/Human-wt/HUN/182-02/2001/G8P[14]. The whole genome constellation (G8-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3) of this strain was shared with another Hungarian zoonotic G8P[14] strain, RVA/Human-wt/HUN/BP1062/2004/G8P[14], although phylogenetic analyses revealed the two rotaviruses likely had different progenitors. Overall, our findings indicate that human G8P[14] rotavirus detected in Hungary in the past originated from independent zoonotic events. Further studies are needed to assess the public health risk associated with infections by various animal rotavirus strains.

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.

Lineage diversification, homo- and heterologous reassortment and recombination shape the evolution of chicken orthoreoviruses.

The near complete genome sequences of ten field avian orthoreovirus (ARV) strains collected from young chicken between 2002 and 2011 in Hungary have been determined in order to explore the genetic diversity and evolutionary mechanisms affecting ARVs in this region. Sequence analyses and phylogenetic calculations revealed similar geographic distribution and distinct evolution in case of eight studied strains that were closely related to the recently described Hungarian strain T1781. The remaining two strains showed the highest similarity with the US origin AVS-B. The topology of the phylogenetic trees of certain segments was affected by several potential homologous reassortment events between strains of Hungarian, Chinese and US origin. Analyzing the µB gene a possible heterologous reassortment event was identified in three Hungarian strains. Recombination events were detected in as much as a dozen cases implying that beside point mutations and reassorment this mechanism also plays an important role in the diversification of ARVs. All these mechanisms in concert may explain the reduced effectiveness of immunization using commercial vaccine strains.

Whole genome sequencing and phylogenetic characterization of brown bullhead (Ameiurus nebulosus) origin ranavirus strains from independent disease outbreaks.

Ranaviruses are emerging pathogens associated with high mortality diseases in fish, amphibians and reptiles. Here we describe the whole genome sequence of two ranavirus isolates from brown bullhead (Ameiurus nebulosus) specimens collected in 2012 at two different locations in Hungary during independent mass mortality events. The two Hungarian isolates were highly similar to each other at the genome sequence level (99.9% nucleotide identity) and to a European sheatfish (Silurus glanis) origin ranavirus (ESV, 99.7%-99.9% nucleotide identity). The coding potential of the genomes of both Hungarian isolates, with 136 putative proteins, were shared with that of the ESV. The core genes commonly used in phylogenetic analysis of ranaviruses were not useful to differentiate the two brown bullhead ESV strains. However genome-wide distribution of point mutations and structural variations observed mainly in the non-coding regions of the genome suggested that the ranavirus disease outbreaks in Hungary were caused by different virus strains. At this moment, due to limited whole genome sequence data of ESV it is unclear whether these genomic changes are useful in molecular epidemiological monitoring of ranavirus disease outbreaks. Therefore, complete genome sequencing of further isolates will be needed to identify adequate genetic markers, if any, and demonstrate their utility in disease control and prevention.

Taxonomy of the order Mononegavirales: update 2016.

In 2016, the order Mononegavirales was emended through the addition of two new families (Mymonaviridae and Sunviridae), the elevation of the paramyxoviral subfamily Pneumovirinae to family status (Pneumoviridae), the addition of five free-floating genera (Anphevirus, Arlivirus, Chengtivirus, Crustavirus, and Wastrivirus), and several other changes at the genus and species levels. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Neuroinvasive influenza virus A(H5N8) in fattening ducks, Hungary, 2015.

Highly pathogenic avian influenza (HPAI) A virus H5N8 was detected in far east Asian countries during 2014 and emerged in late 2014 in European countries. Hungary reported a HPAI A(H5N8) outbreak during late winter of 2015 at a Pekin duck fattening facility. Epidemiologic monitoring was extended to holdings in neighboring areas and nearby habitats used by wild birds but failed to identify the source of infection. In addition to respiratory symptoms, the affected birds showed lethargy and neuronal signs, including torticollis. Consistent with this finding, influenza A virus antigen was detected in large quantity in the brain. Molecular analysis of the identified strain showed very close genetic relationship (and >99% nucleotide sequence identity) with co-circulating HPAI A(H5N8) strains. A number of unique or rarely detected amino acid changes was detected in the HA (T220I, R512G), the M2 (I39M), the NA (T211I), the NS1 (P85T), and the PB2 (I261V) proteins of the Hungarian strain. Further studies are needed to demonstrate whether any of these mutations can be linked to neuroinvasiveness and neurovirulence in ducks.

Development of a consensus reverse transcription PCR assay for the specific detection of tortoise picornaviruses.

Picornaviruses (PVs) of different terrestrial tortoise species, previously designated as Virus "X," have been frequently detected from various tissues by virus isolation in Terrapene heart cell culture as the preferred laboratory method for diagnosis. Here, we describe the development of 2 diagnostic reverse transcription (RT)-PCR-based assays for the identification and characterization of tortoise PVs belonging to the tentative genus Topivirus To test the novel diagnostic systems, PVs were isolated from swab and tissue samples collected in Germany, Italy, and Hungary between 2000 and 2013. All 25 tested isolates gave positive results with both novel consensus primer sets. Sequencing of the amplified products confirmed that all studied viruses were members of the new proposed genus Topivirus Phylogenetic analyses clearly distinguished 2 lineages within the genus. Based on sequence analysis, no association was observed between the geographic distribution and genetic relatedness. Furthermore, no strict host specificity was indicated. The PCR-based diagnosis may provide a time-saving and sensitive method to detect tortoise PVs, and evaluation of PV presence in these animals may help control virus spread.

Complete Genome Sequence of a Genotype G23P[37] Pheasant Rotavirus Strain Identified in Hungary.

We investigated the genomic properties of a rotavirus A strain isolated from diarrheic pheasant poults in Hungary in 2015. Sequence analyses revealed a shared genomic constellation (G23-P[37]-I4-R4-C4-M4-A16-N10-T4-E4-H4) and close relationship (range of nucleotide sequence similarity: VP2, 88%; VP1 and NSP4, 98%) with another pheasant rotavirus strain isolated previously in Germany.

Whole genome characterization of a chelonian orthoreovirus strain identifies significant genetic diversity and may classify reptile orthoreoviruses into distinct species.

In this study we report the sequence and phylogenetic characterization of an orthoreovirus strain, CH1197/96, isolated from a spur-thighed tortoise (Testudo graeca) on chicken embryo fibroblast cells. The 23,957 bp long genome sequence was obtained by combined use of semiconductor and capillary sequencing. Although the genomic characterization showed that the virus was most similar to the bush viper reovirus strain, 47/02, and in phylogenies performed with all segments the two strains formed a monophyletic group, the nucleotide (48.4-70.3%) and amino acid (39.2-80.7%) sequence identity values were moderate between the two reptile origin reoviruses. Based on our results and existing classification criteria for the genus Orthoreovirus, the tortoise reovirus strain CH1197/96 might be the first representative of a novel reptilian origin Orthoreovirus species.

Phylogenetic analysis of a novel reassortant orthoreovirus strain detected in partridge (Perdix perdix).

Avian orthoreoviruses cause various diseases in wild birds and domesticated poultry. In this study we report the detection and genomic characterization of a partridge (Perdix perdix) origin reovirus strain, D1007/2008. The virus was isolated on cell culture from acute pneumonia and infra-orbital sinusitis. The 23,497 nucleotide long genome sequence was obtained by combined use of semiconductor and capillary sequencing. Sequence and phylogenetic analyses showed that the partridge reovirus strain was related to orthoreoviruses of gallinaceous birds. In fact, five (λB, λC, µB, σC, σNS) and one (σB) out of 10 genes clustered definitely with turkey or chicken origin orthoreoviruses, respectively, whereas in the λA, µA, µNS and σA phylogenies a more distant genetic relationship was observed. Our data indicate that the identified reovirus strain is composed of a mixture of chicken and turkey orthoreovirus related alleles. This finding implies that partridges may serve as natural reservoirs for orthoreoviruses of domesticated poultry.

Genome Sequences of Three Turkey Orthoreovirus Strains Isolated in Hungary.

We have investigated the genomic properties of three turkey reovirus strains-19831M09, D1246, and D1104-isolated in Hungary in 2009. Sequence identity values and phylogenetic calculations indicated genetic conservativeness among the studied Hungarian strains and a close relationship with strains isolated in the United States.

Zoonotic transmission of rotavirus: surveillance and control.

Group A rotavirus (Rotavirus A, RVA) is the main cause of acute dehydrating diarrhea in humans and numerous animal species. RVA shows vast diversity and a variety of human strains share genetic and antigenic features with animal origin RVA strains. This finding suggests that interspecies transmission is an important mechanism of rotavirus evolution and contributes to the diversity of human RVA strains. RVA is responsible for half a million deaths and several million hospitalizations worldwide. Globally, two rotavirus vaccines are available for routine use in infants. These vaccines show a great efficacy profile and induce protective immunity against various rotavirus strains. However, little is known about the long-term evolution and epidemiology of RVA strains under selective pressure related to vaccine use. Continuous strain surveillance in the post-vaccine licensure era is needed to help better understand mechanisms that may affect vaccine effectiveness.

Unique genomic organization of a novel Avipoxvirus detected in turkey (Meleagris gallopavo).

Avipoxviruses are emerging pathogens affecting over 200 bird species worldwide. Genetic characterization of avipoxviruses is performed by analysis of genomic regions encoding the 4b and DNA polymerase. Whole genome sequence data are limited to a few avipoxvirus isolates. Based on phylogenetic analysis three major genetic clades are distinguished. In this study we report a novel avipoxvirus strain causing skin lesions in domestic turkey. The virus was identified in Hungary during 2011 in a flock of turkey vaccinated against avipoxvirus infection. The genome of the isolated strain, TKPV-HU1124/2011, was uniquely short (∼188.5kbp) and was predicted to encode reduced number of proteins. Phylogenetic analysis of the genes encoding the 4b and DNA polymerase separated TKPV-HU1124/2011 from other turkey origin avipoxviruses and classified it into a new genetic clade. This study permits new insight into the genetic and genomic heterogeneity of avipoxviruses and pinpoints the importance of strain diversity in vaccine efficacy.

Coding-complete sequencing classifies parrot bornavirus 5 into a novel virus species.

In this study, we determined the sequence of the coding region of an avian bornavirus detected in a blue-and-yellow macaw (Ara ararauna) with pathological/histopathological changes characteristic of proventricular dilatation disease. The genomic organization of the macaw bornavirus is similar to that of other bornaviruses, and its nucleotide sequence is nearly identical to the available partial parrot bornavirus 5 (PaBV-5) sequences. Phylogenetic analysis showed that these strains formed a monophyletic group distinct from other mammalian and avian bornaviruses and in calculations performed with matrix protein coding sequences, the PaBV-5 and PaBV-6 genotypes formed a common cluster, suggesting that according to the recently accepted classification system for bornaviruses, these two genotypes may belong to a new species, provisionally named Psittaciform 2 bornavirus.