Recombination analysis was carried out using RDP4 software, utilizing representative complete genome sequences of gyroviruses. Pairwise identity values were calculated using SDTv1.2 and Geneious Prime software. Phylogenetic analysis of the VP1 coding region was performed by the maximum-likelihood method (GTR+G model, 1000 bootstrap replicates), using MEGA6 software. The ORFs of the assembled viral genome sequence were predicted using the ORF Finder tool ( ). The PCR thermal profile consisted of an initial denaturation step at 95 ☌ for 3 min, 45 cycles of amplification with steps of 95 ☌ for 30 s, 53 ☌ for 30 s, and 72 ☌ for 1 min, followed by a final elongation step at 72 ☌ for 10 min. ![]() Twenty μl of PCR reaction mixture, prepared for amplification of this region, contained 250 μM dNTPs, 250 nM primers (GyV3-F1 and GyV3-R1 GyV3-F2 and GyV3-R2 ), 1x DreamTaq Buffer, 0.625 U of DreamTaq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and 1 μL of the nucleic acid. After de novo assembly, a missing sequence in the non-translated region (NTR) of the genome was obtained by direct sequencing of two PCR products. The sequences were edited and aligned using AliView and Geneious Prime software. Sequence reads were assembled de novo into contigs using Geneious Prime software v.2020.2.4 (Biomatters, Auckland, New Zealand) and were checked by BLAST analysis. The trimmed reads were submitted for taxonomic classification to the Kaiju web server. After amplification by random RT-PCR, a cDNA library was prepared for next-generation sequencing on an Illumina NextSeq™ 500 platform according to a previously described protocol. Nucleic acid was extracted using a NucleoSpin RNA Virus Kit (Macherey-Nagel, Düren, Germany). The homogenate was centrifuged (10,000 × g for 5 min) and filtered through a 0.45-µm PES filter. Approximately 100 mg of specimen was homogenized in phosphate-buffered saline (PBS), using a TissueLyzer LT instrument (QIAGEN, Hilden, Germany). ![]() The bird succumbed to poult enteritis and mortality syndrome in 2017 on a pheasant farm in Hungary. In this study, a mixed organ sample (intestine, brain, heart, liver, and spleen) of a common pheasant ( Phasianus colchicus) was subjected to metagenomic analysis. Although there is no evidence that these viruses are pathogenic to their respective hosts, a recent study described gyrovirus 3 (GyV3, species Gyrovirus homsa 1) to be a multi-host pathogen, infecting mice and chickens. Other gyroviruses have been identified in organ and fecal specimen of domestic and wild birds (e.g., chicken, Gallus gallus northern fulmar, Fulmarus glacialis crested screamer, Chauna torquata ashy storm petrel, Hydrobates homochroa ferruginous-backed antbird, Myrmoderus ferrugineus white-plumed antbird, Pithys albifrons grey teal, Anas gracilis pigeon, Columba livia Pekin duck, Anas platyrhynchos), mammals (human, Homo sapiens domestic cat, Felis catus ferret, Mustela putorius furo) and reptiles (king rat snake, Elaphe carinata). Chicken anemia virus (CAV) is an immunosuppressive agent of chickens that can cause growth and feathering abnormalities, as well as anemia, and predisposes the host to secondary infections. Gyroviruses are classified into 10 species, including Chicken anemia virus and nine additional species: Gyrovirus fulgla 1, Gyrovirus galga 1 and 2, Gyrovirus homsa 1, 2, 3 and 4, Gyrovirus hydho1, and Gyrovirus myferr 1.
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