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.

Structural similarity of human papillomavirus E4 and polyomaviral VP4 exhibited by genomic analysis of the common kestrel (Falco tinnunculus) polyomavirus.

Polyomaviruses are widely distributed viruses of birds that may induce developmental deformities and internal organ disorders primarily in nestlings. In this study, polyomavirus sequence was detected in kidney and liver samples of a common kestrel (Falco tinnunculus) that succumbed at a rescue station in Hungary. The amplified 5025 nucleotide (nt) long genome contained the early (large and small T antigen, LTA and STA) and late (viral proteins, VP1, VP2, VP3) open reading frames (ORFs) typical for polyomaviruses. One of the additional putative ORFs (named VP4) showed identical localization with the VP4 and ORF-X of gammapolyomaviruses, but putative splicing sites could not be found in its sequence. Interestingly, the predicted 123 amino acid (aa) long protein sequence showed the highest similarity with human papillomavirus E4 early proteins in respect of the aa distribution and motif arrangement implying similar functions. The LTA of the kestrel polyomavirus shared <59.2% nt and aa pairwise identity with the LTA sequence of other polyomaviruses and formed a separated branch in the phylogenetic tree among gammapolyomaviruses. Accordingly, the kestrel polyomavirus may be the first member of a novel species within the Gammapolyomavirus genus, tentatively named Gammapolyomavirus faltin.

Deep Sequencing of Porcine Reproductive and Respiratory Syndrome Virus ORF7: A Promising Tool for Diagnostics and Epidemiologic Surveillance.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major concern worldwide. Control of PRRSV is a challenging task due to various factors, including the viral diversity and variability. In this study, we evaluated an amplicon library preparation protocol targeting the ORF7 region of both PRRSV species, Betaarterivirus suid 1 and Betaarterivirus suid 2. We designed tailed primers for a two-step PCR procedure that generates ORF7-specific amplicon libraries suitable for use on Illumina sequencers. We tested the method with serum samples containing common laboratory strains and with pooled serum samples (n = 15) collected from different pig farms during 2019-2021 in Hungary. Testing spiked serum samples showed that the newly designed method is highly sensitive and detects the viral RNA even at low copy numbers (corresponding to approx. Ct 35). The ORF7 sequences were easily assembled even from clinical samples. Two different sequence variants were identified in five samples, and the Porcilis MLV vaccine strain was identified as the minor variant in four samples. An in-depth analysis of the deep sequencing results revealed numerous polymorphic sites along the ORF7 gene in a total of eight samples, and some sites (positions 12, 165, 219, 225, 315, 345, and 351) were found to be common in several clinical specimens. We conclude that amplicon deep sequencing of a highly conserved region of the PRRSV genome could support both laboratory diagnosis and epidemiologic surveillance of the disease.

Genomic Epidemiology and Evolution of Fowl Adenovirus 1.

Fowl adenovirus 1 (FAdV-1) is the main cause of gizzard erosion in chickens. Whole genome sequencing and sequence analyses of 32 FAdV-1 strains from a global collection provided evidence that multiple recombination events have occurred along the entire genome. In gene-wise phylogenies, only the adenoviral pol gene formed a tree topology that corresponded to whole genome-based phylogeny. Virus genetic features that were clearly connected to gizzard erosion were not identified in our analyses. However, some genome variants tended to be more frequently identified from birds with gizzard erosion and strains isolated from healthy birds or birds with non-specific pathologies tended to form common clusters in multiple gene phylogenies. Our data show that the genetic diversity is greater, and the evolutionary mechanisms are more complex within FAdV-1 than previously thought. The implications of these findings for viral pathogenesis and epidemiology await further investigation.

Mechanisms of circovirus immunosuppression and pathogenesis with a focus on porcine circovirus 2: a review.

Certain pathogens, due to their adverse effects on the immune reaction, aggravate the course of concomitant heterologous infections. Here we summarize mechanisms by which circoviruses, including the most studied porcine circovirus 2, and other mammalian and avian circoviruses, trigger their own replication and confound the hosts' immune response. At different stages of infection, from latent state to disease induction, these viruses markedly influence the cellular signaling pathways. Circoviruses have been found to interfere with interferon and proinflammatory cytokine producing and responsive pathways. Apoptotic processes, altered cellular transport and constraint of the mitotic phase all support the viral replication. The cytokine imbalance and lymphocyte depletion, thus the impaired immunity, favors invasion of super- or co-infecting agents, which in concert with circoviruses induce illnesses with increased severity. The information summarized in this review point out the diversity of host and viral factors involved in the mechanisms of disease progression during circovirus infections.

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.

A novel gammapolyomavirus in a great cormorant (Phalacrocorax carbo).

In this study, the complete genome of a novel polyomavirus detected in a great cormorant (Phalacrocorax carbo) was characterized. The 5133-bp-long genome of the cormorant polyomavirus has a genomic structure typical of members of the genus Gammapolyomavirus, family Polyomaviridae, containing open reading frames encoding the large and small tumor antigens, viral proteins 1, 2, and 3, and the X protein. The large tumor antigen of the cormorant polyomavirus shares 45.6-50.4% amino acid sequence identity with the homologous sequences of other gammapolyomaviruses. These data, together with results of phylogenetic analysis, suggest that this cormorant polyomavirus should be considered the first member of a new species within the genus Gammapolyomavirus, for which we propose the name "Phalacrocorax carbo polyomavirus 1".

Novel Circoviruses from Birds Share Common Evolutionary Roots with Fish Origin Circoviruses.

Circoviruses occur in a variety of animal species and are common pathogens of mammalian and avian hosts. In our study internal organ samples of wild birds were processed for screening of circoviral sequences. Two novel viruses were identified and characterized in specimens of a little bittern and a European bee-eater that suffered from wing injuries, were weakened, had liver or kidney failures, and finally succumbed at a rescue station. The 1935 nt and 1960 nt long viral DNA genomes exhibited a genomic structure typical for circoviruses and were predicted to encode replication-associated protein in the viral strand, and a capsid protein in the complementary strand of the replicative intermediate DNA form. The genome of the newly described viruses showed 37.6% pairwise identity with each other and ≤41.5% identity with circovirus sequences, and shared a common branch with fish, human and Weddel seal circoviruses in the phylogenetic tree, implying evolutionary relationship among the ancestors of these viruses. Based on the results the little bittern and European bee-eater circoviruses represent two distinct species of the Circovirus genus, Circoviridae family.

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".

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.

Viral gene expression profile of goose haemorrhagic polyomavirus in susceptible primary cells.

Routine culturing of goose haemorrhagic polyomavirus (GHPV) is cumbersome, and limited data are available about its replication and gene expression profile. In this study, goose embryo fibroblast cells were infected with GHPV for temporal measurement of the viral genome copy number and mRNA levels with quantitative PCR. Accumulation of small and large tumour antigen-encoding mRNAs was detected as early as 9 hours post-infection (hpi), while high level expression of the capsid protein encoding VP1-VP3, and ORF-X mRNAs was first detected at 24 hpi. Elevation of GHPV genome copy number was noted at 48 hpi. The results indicate that the gene expression profile of GHPV is similar to that described for mammalian polyomaviruses.RESEARCH HIGHLIGHTS GHPV was propagated in culture of primary goose embryo fibroblast cells.The transcription commenced before the onset of viral DNA replication.The transcription patterns of GHPV and mammalian polyomaviruses were comparable.

Genomic Epidemiology and Evolution of Duck Hepatitis A Virus.

Duck hepatitis A virus (DHAV), an avian picornavirus, causes high-mortality acute disease in ducklings. Among the three serotypes, DHAV-1 is globally distributed, whereas DHAV-2 and DHAV-3 serotypes are chiefly restricted to Southeast Asia. In this study, we analyzed the genomic evolution of DHAV-1 strains using extant GenBank records and genomic sequences of 10 DHAV-1 strains originating from a large disease outbreak in 2004-2005, in Hungary. Recombination analysis revealed intragenotype recombination within DHAV-1 as well as intergenotype recombination events involving DHAV-1 and DHAV-3 strains. The intergenotype recombination occurred in the VP0 region. Diversifying selection seems to act at sites of certain genomic regions. Calculations estimated slightly lower rates of evolution of DHAV-1 (mean rates for individual protein coding regions, 5.6286 × 10-4 to 1.1147 × 10-3 substitutions per site per year) compared to other picornaviruses. The observed evolutionary mechanisms indicate that whole-genome-based analysis of DHAV strains is needed to better understand the emergence of novel strains and their geographical dispersal.

Genomic Diversity of CRESS DNA Viruses in the Eukaryotic Virome of Swine Feces.

Replication-associated protein (Rep)-encoding single-stranded DNA (CRESS DNA) viruses are a diverse group of viruses, and their persistence in the environment has been studied for over a decade. However, the persistence of CRESS DNA viruses in herds of domestic animals has, in some cases, serious economic consequence. In this study, we describe the diversity of CRESS DNA viruses identified during the metagenomics analysis of fecal samples collected from a single swine herd with apparently healthy animals. A total of nine genome sequences were assembled and classified into two different groups (CRESSV1 and CRESSV2) of the Cirlivirales order (Cressdnaviricota phylum). The novel CRESS DNA viral sequences shared 85.8-96.8% and 38.1-94.3% amino acid sequence identities for the Rep and putative capsid protein sequences compared to their respective counterparts with extant GenBank record. Data presented here show evidence for simultaneous infection of swine herds with multiple novel CRESS DNA viruses, including po-circo-like viruses and fur seal feces-associated circular DNA viruses. Given that viral genomes with similar sequence and structure have been detected in swine fecal viromes from independent studies, investigation of the association between presence of CRESS DNA viruses and swine health conditions seems to be justified.

Genomic evolution of avian polyomaviruses with a focus on budgerigar fledgling disease virus.

Gammapolyomaviruses may cause serious inflammatory diseases in a broad range of avian hosts. In this study we investigated genomic evolution of and selection constraint acting on avian polyomaviruses (APyVs). Our analyses suggested that goose haemorrhagic polyomavirus (GHPV) evolves more slowly (3.03 × 10-5 s/s/y mean evolutionary rate) than budgerigar fledgling disease virus (BFDV), finch polyomavirus (FPyV) and canary polyomavirus (CaPyV) (1.39 × 10-4 s/s/y, 2.63 × 10-4 s/s/y and 1.41 × 10-4 s/s/y mean evolutionary rate, respectively). In general, purifying selection seems to act on the protein coding regions of APyV genomes, although positive Darwinian selection was also predicted in a few positions (e.g., in the large tumor antigen coding region of BFDV and GHPV and in the capsid protein sequences of BFDV). The importance of these aa changes remains elusive. Overall, a better understanding of adaptive changes in the genome of APyVs requires additional data from various incidental hosts and reservoir species.

Phylogeny of Hungarian EBLV-1 strains using whole-genome sequence data.

European bat lyssavirus 1 (EBLV-1) is a widespread lyssavirus across Europe, whose epizootic cycle is linked to a few bat species. Occasionally, EBLV-1 infection may occur in domestic animals and humans. EBLV-1 can be classified into two subtypes, where subtype EBLV-1a shows a wide geographic distribution between France and Russia whereas subtype EBLV-1b is distributed between Spain and Poland. In this study, we determined the genome sequence of two recent EBLV-1a strains detected in Hungary and analysed their adaptive evolution and phylodynamics. The data set that included 100 EBLV-1 genome sequences identified positive selection at selected sites in genes coding for viral proteins (N, codon 18; P, 141 and 155; G, 244 and 488; L, 168, 980, 1597 and 1754). A major genetic clade containing EBLV-1a isolates from Hungary, Slovakia, Denmark and Poland was estimated to have diverged during the 19th century whereas the divergence of the most recent ancestor of Hungarian and Slovakian isolates dates back to 1950 (time span, 1930 to 1970). Phylogeographic analysis of the EBLV-1a genomic sequences demonstrated strong evidence of viral dispersal from Poland to Hungary. This new information indicates that additional migratory flyways may help the virus spread, a finding that supplements the general theory on a west-to-east dispersal of EBLV-1a strains. Long-distance migrant bats may mediate the dispersal of EBLV-1 strains across Europe; however, structured surveillance and extended genome sequencing would be needed to better understand the epizootiology of EBLV-1 infections in Europe.

Publisher Correction: Occurrence and genetic diversity of CRESS DNA viruses in wild birds: a Hungarian study.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular epidemiology and phylodynamics of goose haemorrhagic polyomavirus.

Goose haemorrhagic polyomavirus (GHPV, or Anser anser polyomavirus 1) is a small dsDNA virus of the Polyomaviridae family. The virus infects the internal organs causing haemorrhagic nephritis and enteritis of geese that may be fatal for goslings. In this study, GHPV positivity was examined in goose and duck samples collected in Hungary between 2005 and 2019. In this period, 384 of the investigated 1,111 specimens were diagnosed as GHPV-positive by PCR assay. Twenty-two GHPV genomes were sequenced and subjected to phylogenetic and evolutionary analysis. Based on the sequence data, the mean evolutionary rates were estimated 6.57 × 10-6 -5.82 × 10-5  s/s/y for both GHPV complete genomes and individual genes, with negative selection acting on each gene. When GHPV VP1 sequences originating from wild birds were also included in the analyses, the nt and aa mutations inflated the substitution rate to 1.54 × 10-4  s/s/y that may imply adaptation of the virus to novel host species. Our data suggested the co-circulation of various GHPV strains in Hungarian goose farms; the source of these may be persistently infected domesticated or migratory wild birds. Detection and characterization of GHPV in wild birds and domestic waterfowls may help to elaborate new strategies for more effective disease control and prevention.

Occurrence and genetic diversity of CRESS DNA viruses in wild birds: a Hungarian study.

Circoviruses, cycloviruses and other circular, replication-associated protein-encoding single stranded (CRESS) DNA viruses have been detected in a variety of animal taxa. In this study, cloacal swab samples (n = 90) were examined for CRESS DNA viruses from 31 wild bird species living at various aquatic sites in Hungary to identify possible reservoirs of viruses pathogenic to domestic poultry. A total of 30 (33.3%) specimens tested positive with pan-CRESS DNA virus specific PCR. Goose circovirus (GoCV), Duck associated cyclovirus 1 (DuACyV-1) and Garrulus glandarius associated circular virus 1 (GgaCV-1) were detected in nine, three and two different bird species, respectively. Selected specimens were subjected to whole genome sequencing. The obtained sequence data revealed conserved gene structure within the identified virus species and detected homologous (within GoCV) and possible heterologous recombination (within DuACyV-1) events. Results presented here provide new information on the genomic diversity and evolution of selected CRESS DNA viruses.

Coding-complete genome sequencing suggests that Newcastle disease virus challenge strain Herts'33 (IVMP) may represent a distinct genotype.

We determined the genomic sequence of a Newcastle disease virus (NDV) line obtained directly from the first NDV isolate, named Herts'33. This strain shared ≤ 90% nucleotide sequence identity with the NDV sequences available in the GenBank database, and formed a distinct branch in a phylogenetic tree. This branch may be considered to represent a separate NDV genotype. Our study indicates that investigation of the genomic sequences of old NDV strains that originated from the early outbreaks of Newcastle disease may alter the phylogenetic grouping of the NDV strains and provide data on the evolution of viral genomes over time.

Phylogenetic Analysis of Lednice Orthobunyavirus.

Lednice virus (LEDV) has been detected in Culex modestus mosquitoes in several European countries within the last six decades. In this study, phylogenetic analyses of the complete genome segments confirm that LEDV belongs to the Turlock orthobunyavirus (Orthobunyavirus, Peribunyaviridae) species and is closely related to Umbre, Turlock, and Kedah viruses.