Molecular detection and characterization of human gyroviruses identified in the ferret fecal virome

Abstract

The recently described novel gyroviruses may infect chickens and/or humans, however, their pathogenic potential is unknown. In our metagenomic investigation we detected many of the novel gyroviruses in the fecal viromes of ferrets with lymph node and organ enlargement. The whole genomic sequences of selected gyrovirus strains showed 90.7-99.4% similarity with homologous reference gyrovirus strains. This study does not elucidate an association between gyrovirus shedding of ferrets and the observed background disease; however, it provides evidence for genetic diversity within gyroviruses and raises the possibility that pet ferrets may transmit gyroviruses to heterologous hosts, e.g. humans.Manuscript Click here to download Manuscript: 20140623_MS_Feher.doc Click here to view linked References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 Several new members of the genus Gyrovirus (family Circoviridae) have been described during the past three years. Chicken anaemia virus (CAV) causes anaemia, bone marrow atrophy and severe immunosuppression in poultry, and represents the prototype species within the genus. Newly described members include human gyrovirus (HGyV), avian gyrovirus 2 (AGV2), GyV3, GyV4,. Of interest, some of these new gyrovirus types have been identified in human skin (HGyV), blood (HGyV) and stool (all gyroviruses) specimens, although the etiologic role of these viruses in a human disease has not been revealed [1][2][3][4][5][6][7]. Furthermore, in addition to CAV, AGV2, HGyV and GyV4 have been also detected in chicken serum, meat and skin [1][2][3].In this study, 23 diarrheic stool specimens were collected from 20 pet ferrets (Mustela putorius furo) housed in a shelter. The animals had a background disease characterized by lymph node and spleen enlargement.The majority of samples (21 samples of 18 ferrets) were subjected to viral metagenomics. In brief, the genomic RNA was extracted using the Viral RNA Mini kit (QIAGEN) according to the manufacturer's instructions. The RNA sample was denatured at 97°C for 5 min in the presence of 10 µM random hexamer tailed by a common PCR primer sequence [8]. Reverse transcription reaction mixture containing 400 µM dNTPs, 1X AMV RT buffer and 1 U AMV reverse transcriptase (Promega) was added and then incubated at 42°C for 45 min. 5 µl cDNA was amplified in a final reaction volume of 50 µl including 500 µM PCR primers, 200 µM dNTP mixture, 1.5 mM MgCl2, 1X Taq DNA polymerase buffer and 0.5 U Taq DNA polymerase (Thermo Scientific).The reaction conditions consisted of a denaturation step at 95°C for 3 min, 40 cycles of amplification (95°C for 30 sec, 48°C for 30 sec, 72°C for 2 min) and a final extension step at 72°C for 8 min.Enzymatic fragmentation was carried out from 100 ng of the amplified cDNA using the reagents of the surprising in the fecal specime...

A new gyrovirus in human feces

“…Gyrovirus CAV is a very frequent chicken infection, and CAV DNA is commonly seen in human feces [9]. Gyrovirus DNA has also been reported in the feces of carnivore, possibly from the consumption of chicken or other birds [33, 34]. The detection of diverse gyrovirus DNA in human feces may reflect recent consumption of infected chicken, a frequent source of diarrhea-causing bacterial infections [35, 36], rather than active gyrovirus replication in human cells.…”

“…Gyrovirus DNA may therefore survive transit through the human gut without playing any role in this patient’s diarrhea [38, 39]. Despite the detection of gyrovirus DNA detection in human blood, on human skin, and in feces of human and carnivorous mammals [6–8, 33, 34], gyrovirus replication in non-avian species such as mammals remains to be conclusively demonstrated.…”

Sequence and phylogenetic analysis identifies a putative novel gyrovirus 3 genotype in ferret feces

“…However, contrary to our previous study [8], this approach worked only when the modified nucleotide, 7-deazadGTP, was incorporated in the back-to-back PCR assay. The PCR mixture contained 50 µM of dATP, dCTP, dGTP and dTTP, 50 µM of 7-deaza-dGTP (New England Biolabs), 200 nM of each primers, 1X Phusion Green HF Buffer and 0.3 U Phusion DNA polymerase (Thermo Scientific).…”

“…The PCR mixture contained 50 µM of dATP, dCTP, dGTP and dTTP, 50 µM of 7-deaza-dGTP (New England Biolabs), 200 nM of each primers, 1X Phusion Green HF Buffer and 0.3 U Phusion DNA polymerase (Thermo Scientific). The cycling profile was basically the same as described elsewhere for other GyVs [8], although we used 54 ºC primer annealing temperature in this experiment. The ~2.3 kb long PCR products obtained by various combinations of the back-to-back primers were purified (Geneaid) from gel slices and then directly sequenced on an ABI PRISM® 3100-Avant Genetic Analyzer using the BigDye Terminator v1.1 Cycle Sequencing Kit (Life Technologies), with the addition of 2.5 µM of 7-deaza-dGTP to the sequencing reactions.…”

“…In a previous study traces of CAV, HGyV/AGV2, GyV3 and GyV4 were detected in the fecal samples of pet ferrets (Mustela putorius furo) by viral metagenomics and by diagnostic PCRs [8]; however, discrepancy in the positivity rate for GyV3 and GyV4 was seen when the results obtained by the two methods were merged, probably due to the extensive sequence variability. A partial fragment of a distant relative of GyV3 was also detected by deep sequencing, but could not be fully analysed because of issues in whole genome amplification and sequencing.…”