A Snapshot on the Genomic Epidemiology of Turkey Reovirus Infections, Hungary.

Reovirus infections in turkeys are associated with arthritis and lameness. Viral genome sequence data are scarce, which makes an accurate description of the viral evolution and epidemiology difficult. In this study, we isolated and characterized turkey reoviruses from Hungary. The isolates were identified in 2016; these isolates were compared with earlier Hungarian turkey reovirus strains and turkey reoviruses isolated in the 2010s in the United States. Gene-wise sequence and phylogenetic analyses identified the cell-receptor binding protein and the main neutralization antigen, σC, to be the most conserved. The most genetically diverse gene was another surface antigen coding gene, µB. This gene was shown to undergo frequent reassortment among chicken and turkey origin reoviruses. Additional reassortment events were found primarily within members of the homologous turkey reovirus clade. Our data showed evidence for low variability among strains isolated from independent outbreaks, a finding that suggests a common source of turkey reoviruses in Hungarian turkey flocks. Given that commercial vaccines are not available, identification of the source of these founder virus strains would permit a more efficient prevention of disease outbreaks before young birds are settled to fattening facilities.

Marked Genotype Diversity among Reoviruses Isolated from Chicken in Selected East-Central European Countries.

The concern that the vaccines currently used against Avian orthoreovirus (ARV) infections are less efficient in the field justifies the need for the close monitoring of circulating ARV strains. In this study, we collected necropsy samples from various chicken breeds and tested for ARV by virus isolation, RT-PCR assay and sequence analysis. ARVs were isolated from birds showing runting-stunting syndrome, uneven growth, lameness or increased mortality, with relative detection rates of 38%, 35%, 6% and 25%, respectively. Partial σC gene sequences were determined for nearly 90% of ARV isolates. The isolates could be classified into one of the major genetic clusters. Interestingly, cluster 2 and cluster 5 were isolated from vaccinated broiler breeders, while clusters 1 to 4 were isolated from unvaccinated broilers. The isolates shared less than 75% amino acid identities with the vaccine strains (range, 44.3-74.6%). This study reaffirms the global distribution of the major genetic clusters of ARVs in chicken. The diversity of ARV strains isolated from unvaccinated broilers was greater than those detected from vaccinated animals, however, the relative importance of passive and active immunity on the selection of novel strains in different chicken breeds needs to be better understood.

Genetic diversity among reptilian orthoreoviruses isolated from pet snakes and lizards.

Reovirus infections in reptiles are frequently detected and associated with various clinical diseases; yet, our knowledge about their genetic diversity and evolutionary relationships remains limited. In this study, we characterize at the genomic level five reptile origin orthoreovirus strains isolated from exotic snakes and lizards in Hungary and Germany. The genomic organization of the study strains was similar to that of the representative strains of reptile origin reoviruses belonging to species Reptilian orthoreovirus and Testudine orthoreovirus. Additionally, all five study strains clustered with the bush viper origin reference Reptilian orthoreovirus strain, 47/02. The nucleotide sequence divergence among strains fell from 56.64 to 99.36%. Based on genome segment constellations two well separated groups were observed, which may represent two genetic lineages of reptilian orthoreoviruses we tentatively referred here as genogroups, classifying two squamata origin strains with available whole genome sequences into genogroup I (GGI) and four strains into genogroup II (GGII). The representative GGI and GGII Reptilian orthoreovirus strains are characterized by moderate-to-high nucleotide and amino acid similarities within genogroups (range, 69.45 to 99.36% and 74.64 to 100.00%), whereas lower nucleotide and amino acid similarities (range, 56.64 to 77.24% and 54.53 to 93.85%) and different structures of the bicistronic S1 segment were found between genogroups. Further studies are needed to explore the genomic diversity among reptilian reoviruses of squamata origin; this would be critical to establish a robust classification system for these viruses and to see if interaction among members of distinct lineages may result in viable progenies with novel genetic features.

Animal Models Used in Monkeypox Research.

Monkeypox is an emerging zoonotic disease with a growing prevalence outside of its endemic area, posing a significant threat to public health. Despite the epidemiological and field investigations of monkeypox, little is known about its maintenance in natural reservoirs, biological implications or disease management. African rodents are considered possible reservoirs, although many mammalian species have been naturally infected with the monkeypox virus (MPXV). The involvement of domestic livestock and pets in spillover events cannot be ruled out, which may facilitate secondary virus transmission to humans. Investigation of MPXV infection in putative reservoir species and non-human primates experimentally uncovered novel findings relevant to the course of pathogenesis, virulence factors and transmission of MPXV that provided valuable information for designing appropriate prevention measures and effective vaccines.

Candidate 'Avian orthoreovirus B': An emerging waterfowl pathogen in Europe and Asia?

A fusogenic virus was isolated from a flock of breeder Pekin ducks in 2019, Hungary. The affected flock experienced a marked decrease in egg production. Histopathological lesions were seen in the oviduct and in the lungs of birds sent for diagnostic investigation. The fusogenic agent was characterized as an orthoreovirus by viral metagenomics. The assembled viral genome was composed of 10 genomic segments and was 23,433 nucleotides (nt) in length. The study strain, designated Reo/HUN/DuckDV/2019, shared low-to-medium gene-wise sequence identity with avian orthoreovirus strains from galliform and anseriform birds (nt, 38.90%-72.33%) as well as with representative strains of neoavian orthoreoviruses (nt, 40.07%-68.23%). On the contrary, the study strain shared 86.48%-95.01% pairwise nt sequence identities with recent German and Chinese reovirus isolates, D2533/6 and Ych, respectively. Phylogenetic analysis clustered all three unusual waterfowl pathogens on a monophyletic branch, indicating a common evolutionary origin of Reo/HUN/DuckDV/2019 with these enigmatic orthoreoviruses described over the past few years. The finding that a candidate new orthoreovirus species, tentatively called Avian orthoreovirus B, was isolated in recent years in Europe and Asia in moribund ducks seems an alarming sign that needs to be better evaluated by extending laboratory diagnosis of viral pathogens in countries where the waterfowl industry is important.

Identification of the main genetic clusters of avian reoviruses from a global strain collection.

Introduction: Avian reoviruses (ARV), an important pathogen of poultry, have received increasing interest lately due to their widespread occurrence, recognized genetic diversity, and association to defined disease conditions or being present as co-infecting agents. The efficient control measures require the characterization of the available virus strains. Methods: The present study describes an ARV collection comprising over 200 isolates from diagnostic samples collected over a decade from 34 countries worldwide. One hundred and thirty-six ARV isolates were characterized based on σC sequences. Results and discussion: The samples represented not only arthritis/tenosynovitis and runting-stunting syndrome, but also respiratory symptoms, egg production problems, and undefined disease conditions accompanied with increased mortality, and were obtained from broiler, layer or breeder flocks. In 31 percent of the cases other viral or bacterial agents were demonstrated besides ARV. The most frequent co-infectious agent was infectious bronchitis virus followed by infectious bursal disease virus and adenoviruses. All isolates could be classified in one of the major genetic clusters, although we observed marked discrepancies in the genotyping systems currently in use, a finding that made genotype assignment challenging. Reovirus related clinical symptoms could not be unequivocally connected to any particular virus strains belonging to a specific genetic group, suggesting the lack of strict association between disease forms of ARV infection and the investigated genetic features of ARV strains. Also, large genetic differences were seen between field and vaccine strains. The presented findings reinforce the need to establish a uniform, widely accepted molecular classification scheme for ARV and further, highlight the need for ARV strain identification to support more efficient control measures.

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 Parvovirus Related to Primate Bufaviruses in Dogs.

A novel protoparvovirus species, related genetically to human bufaviruses, was identified in dogs with respiratory signs. The canine bufavirus was distantly related to the well-known canine protoparvovirus, canine parvovirus type 2, sharing low amino acid identities in the nonstructural protein 1 (40.6%) and in the capsid protein 1 (33.4%). By screening collections of fecal, nasal, and oropharyngeal samples obtained from juvenile dogs (<1 year of age), canine bufavirus DNA appeared as a common component of canine virome. The virus was common in the stool samples of dogs with or without enteric disease and in the nasal and oropharyngeal swab samples of dogs with respiratory signs. However, the virus was not detected in nasal and oropharyngeal swab samples from animals without clinical signs.

Pathogenic potential and virulence genotypes of intestinal and faecal isolates of porcine post-weaning enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) are frequent causes of diarrhoea in infants and in young mammals by inducing attaching effacing (AE) lesions of the intestinal epithelium. EPEC bacteria have also been implicated in cases of porcine post-weaning diarrhoea but their pathogenicity for conventional weaned pigs remains less elucidated. This present study investigates differences in pathogenic potential and virulence genotypes of intestinal and faecal isolates of EPEC from newly-weaned pigs. For this we inoculated ligated ileal loops of four weeks old weaned pigs to assess EPEC adherence to enterocytes by histology and immunohistology. Virulence gene patterns were identified by using a PCR-microarray. Intestinal EPEC isolates of sero-/intimin types O45:H11:eae-ß, O49:NM:eae-ß, O84:H7:eae-γ, and O123:H11:eae-ß formed adherent microcolonies of EPEC with AE lesions on ileal villi more frequently than faecal isolates of O28:H28:eae-NT, O108:H9:eae-ß, O145:H28:eae-γ and O157:H2:eae-ß (p≤0.05). The PCR-array analysis of both groups detected all together 25 virulence genes of LEE (Locus of Enterocyte Effacement), and of non-LEE pathogenicity islands, of plasmids and phages characteristic to EPEC. Intestinal isolates carried significantly more virulence genes than faecal isolates (p≤0.05). Intestinal isolates possessed efa1, lpfA, and tsh genes most likely contributing to enterocyte adhesion while faecal isolates did not carry these genes (p≤0.05). Overall, the ileal loop model in weaned pigs combined with virulence genotyping PCR-array indicated a greater pathogenic potential of intestinal isolates over faecal isolates of porcine post-weaning EPEC. Differing virulence genotypes of the intestinal and faecal isolates as demonstrated here suggests dynamic evolutionary events within the population of porcine EPEC.

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.

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.

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.

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.

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.

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.