A viral metagenomic approach on a non-metagenomic experiment: Mining next generation sequencing datasets from pig DNA identified several porcine parvoviruses for a retrospective evaluation of viral infections

Abstract

Shot-gun next generation sequencing (NGS) on whole DNA extracted from specimens collected from mammals often produces reads that are not mapped (i.e. unmapped reads) on the host reference genome and that are usually discarded as by-products of the experiments. In this study, we mined Ion Torrent reads obtained by sequencing DNA isolated from archived blood samples collected from 100 performance tested Italian Large White pigs. Two reduced representation libraries were prepared from two DNA pools constructed each from 50 equimolar DNA samples. Bioinformatic analyses were carried out to mine unmapped reads on the reference pig genome that were obtained from the two NGS datasets. In silico analyses included read mapping and sequence assembly approaches for a viral metagenomic analysis using the NCBI Viral Genome Resource. Our approach identified sequences matching several viruses of the Parvoviridae family: porcine parvovirus 2 (PPV2), PPV4, PPV5 and PPV6 and porcine bocavirus 1-H18 isolate (PBoV1-H18). The presence of these viruses was confirmed by PCR and Sanger sequencing of individual DNA samples. PPV2, PPV4, PPV5, PPV6 and PBoV1-H18 were all identified in samples collected in 1998–2007, 1998–2000, 1997–2000, 1998–2004 and 2003, respectively. For most of these viruses (PPV4, PPV5, PPV6 and PBoV1-H18) previous studies reported their first occurrence much later (from 5 to more than 10 years) than our identification period and in different geographic areas. Our study provided a retrospective evaluation of apparently asymptomatic parvovirus infected pigs providing information that could be important to define occurrence and prevalence of different parvoviruses in South Europe. This study demonstrated the potential of mining NGS datasets non-originally derived by metagenomics experiments for viral metagenomics analyses in a livestock species.

Metagenomics revealing new virus species in farm and pet animals and aquaculture

“…In pigs, several novel viruses are being constantly detected through metagenomic analysis like circovirus PCV3 , parvovirus (strain swine/ Zsana3/2013/HUN) (Hargitai et al, 2016), Parvovirus 7 (Palinski et al, 2016), etc. Porcine parvovirus 2 (PPV2), PPV4, PPV5, PPV6, and porcine bocavirus 1-H18 isolate (PBoV1-H18) (Bovo et al, 2017), first report for Ndumu virus in Uganda (Masembe et al, 2012), 2009 H1N1, pandemic influenza A virus (Greninger et al, 2010), Torque Teno virus, Porcine boca-like virus (Blomstrom et al, 2010), a new variant of Porcine astrovirus (Karlsson et al, 2015), all have been reported by metagenomics approach. Novel genotype of astrovirus 4 in nasal swab of pigs (Padmanabhan and Hause, 2016) has also been reported.…”

Next generation sequencing in livestock species- A Review

“…Recently Bovo et al (2017) demonstrated the potential of NGS dataset mining for viral metagenomics analysis in livestock.…”

A New Prevalent Densovirus Discovered in Acari. Insight from Metagenomics in Viral Communities Associated with Two-Spotted Mite (Tetranychus urticae) Populations

“…Breakthrough techniques in viral metagenomics and mining high throughput sequencing (HGS) datasets have highlighted the extraordinary diversity and persistence of parvoviruses and parvovirus-related sequences in an unexpected array of animals, including invertebrates [2,3,4]. These discoveries support the now generally accepted view that viruses are integral components of the microbiome and participate in the functioning of ecosystems of differing size and complexity, i.e., an individual organism or a population of organisms.…”