Fecal virome analysis of three carnivores reveals a novel nodavirus and multiple gemycircularviruses
© Conceição-Neto et al. 2015
Received: 20 February 2015
Accepted: 30 April 2015
Published: 20 May 2015
More knowledge about viral populations in wild animals is needed in order to better understand and assess the risk of zoonotic diseases. In this study we performed viral metagenomic analysis of fecal samples from three healthy carnivores: a badger (Meles meles), a mongoose (Herpestes ichneumon) and an otter (Lutra lutra) from Portugal.
We detected the presence of novel highly divergent viruses in the fecal material of the carnivores analyzed, such as five gemycircularviruses. Four of these gemycircularviruses were found in the mongoose and one in the badger. In addition we also identified an RNA-dependent RNA polymerase gene from a putative novel member of the Nodaviridae family in the fecal material of the otter.
Together these results underline that many novel viruses are yet to be discovered and that fecal associated viruses are not always related to disease. Our study expands the knowledge of viral species present in the gut, although the interpretation of the true host species of such novel viruses needs to be reviewed with great caution.
With the advent of next generation sequencing techniques, samples from a wide range of animal species have been screened to identify novel viruses and this approach has become the most important tool for early detection and characterization of possible emerging zoonotic agents [1–3]. It is important to monitor these emerging zoonotic agents as they can be responsible for minor or major epidemics worldwide . For example, zoonoses can range from the Middle East respiratory syndrome coronavirus (MERS-CoV), which recently drew a lot of attention worldwide , to the 2009 pandemic H1N1 influenza A virus or to the less publicized Hepatitis E virus . Not only are humans at risk, but also animals can be infected with viruses from other host species, resulting in disease, or further transmission to humans. The severe acute respiratory syndrome (SARS) coronavirus pandemic originated from wildlife, where bats where identified as the reservoir and civets as an intermediate host [7, 8]. A similar example comes from Hendra virus, which also originated from bats, but transmission to humans occurred with horses as an intermediate host, causing severe pneumonia in horses and humans . As such, zoonotic infections can have significant consequences for animal and public welfare. However, little is known about these pathogens before they emerge from unrecognized zoonotic sources and therefore a deeper understanding of the virome of wild animals will allow us to more rapidly identify the host of particular novel zoonotic viruses, and act appropriately to prevent further spread of such viruses.
Fourteen species of wild carnivores can be found in Portugal, often in relative close contact with humans. Only red fox, stone marten, badger, common genet and, more recently, the Egyptian mongoose, are known to have a generalized distribution throughout the country . From these animals we sampled two species, a badger (Meles meles) and an Egyptian mongoose (Herpestes ichneumon). In addition, we sampled a Eurasian otter (Lutra lutra) raised in a zoo, since otters are also widely distributed in Portugal . Currently little is known about the viral communities that populate the gut of these animals. Thus far, Bodewes and colleagues investigated the fecal virome of badgers and an otter from Spain, and found a fecal phlebovirus in an otter . A study by van den Brand and colleagues investigated the virome of badgers in the Netherlands, identifying two novel circularviruses . In Portugal, a study performed by Oliveira and colleagues screened otters for parvoviruses, adenoviruses and parainfluenza virus, however none of the samples had detectable levels of virus . Also in Portugal, another study screened for and identified parvoviruses in genets, badgers and mongooses . As these findings are probably only the tip of the iceberg, we were interested to further explore the viral communities of the gut in widely spread wild species in Portugal and a zoo specimen.
Results and discussion
Identification of five novel gemycircularviruses in the fecal samples from the badger and mongoose
In recent years, ssDNA viruses have been frequently found in fecal samples of a variety of animal species, such as badgers , bats , cows , turkey , rodents [17, 18], chimpanzees , pigs [20–22], New Zealand fur seal  foxes  and recently also in ancient caribou feces . ssDNA viruses are small viruses (1.4-8.5 kb) and can encode as little as two proteins: a capsid and a replication associated protein. Thus far, seven major ssDNA families have been identified based on the host range and type of ssDNA genome. Only the Parvoviridae family encodes for a linear genome, the remaining are circular. The Nanoviridae and Geminiviridae families infect plants, whereas Circoviridae, Parvoviridae and Anelloviridae are known to infect animals. Viruses belonging to the Inoviridae and Microviridae infect bacteria .
Motifs of the Rep from the novel gemycircularviruses and reference gemycircularviruses
Mongoose feces-associated gemycircularvirus a
Mongoose feces-associated gemycircularvirus b
Mongoose feces-associated gemycircularvirus c
Mongoose feces-associated gemycircularvirus d
Badger feces-associated gemycircularvirus
Fecal associated gemycircularvirus 1a
Faecal associated gemycircularvirus 4
Fecal associated gemycircularvirus 5
Cassava-associated circular DNA virus
Dragonfly-associated circular DNA virus-2
Mosquito VEM virus SDBVL G
Identification a partial RNA-dependent RNA polymerase of a nodavirus in the fecal material of the otter
In the three healthy carnivores analyzed, viral sequences belonging to the Caudovirales order of bacteriophages were also detected, as previously reported . Furthermore, our study showed that even healthy wildlife seems to harbor many divergent viral communities that deserve to be explored further to expand our current knowledge and databases. From the limited data available from the fecal virome studies from an otter (Lutra lutra) and badger (Meles meles) of Bodewes and van der Brand [12, 13], the virus families discovered in our study are completely different, possibly resulting from different diets, or indicating a large unexplored area of the ‘virome space’. Also the fact that the viruses discovered are highly different from viruses available in databases, might explain the difficulty in finding viruses by regular PCR screening, as previously reported . These novel viruses reported in this study are likely derive from the diet, as the Nodavirus from the otter is likely to be from fish and the gemycircularviruses from insects, which are part of the animals diets. Viral discovery can be challenging because novel viruses, as seen in this study, can be quite divergent and their classification and true host determination can be difficult. In this regard, replication associated proteins have shown to be conserved, and the best strategy to create alignments. It is very interesting to see the ubiquity of different circular virus species found nowadays due to the availability of next-generation sequencing. Screening of larger groups of animals and species will help to increase our knowledge of viruses circulating in wild animals.
Fecal samples were collected from a badger (Meles meles), a mongoose (Herpestes ichneumon) from a rescue center and an Eurasian otter (Lutra lutra) from a zoo upon their arrival in 2011 at the wildlife center “Parque Biológico da Serra da Lousã” in Coimbra district, Portugal. This collection was part of the quarantine assessment program applied to newly introduced animals in the center. Samples were kept at −80 °C until further processing.
Ten percent fecal suspensions were homogenized for 1 min at 3000 rpm with a MINILYS homogenizer (Bertin Technologies) and filtered consecutively through 100 μm, 10 μm and 0.8 μm membrane filters (Millipore) for 30 s at 1250 g. The filtrate was then treated with a homemade buffer (1 M Tris, 100 mM CaCl2 and 30 mM MgCl2) and a cocktail of Benzonase (Novagen) and Micrococcal Nuclease (New England Biolabs) at 37 °C for 2 h to digest free-floating nucleic acids. RNA and DNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer’s instructions but without addition of carrier RNA to the lysis buffer. First and second strand synthesis and random PCR amplification for 25 cycles were performed using a slightly modified Whole Transcriptome Amplification (WTA) Kit procedure (Sigma-Aldrich), with a denaturation temperature of 95 °C instead of 72 °C to allow for the denaturation of dsDNA and dsRNA. This modification leads to the amplification of both RNA and DNA. A size selection after library synthesis was performed using a 0.7 ratio of Agencourt AMPure XP beads (Beckman Coulter, Inc.). WTA products were purified with MSB Spin PCRapace spin columns (Stratec) and were prepared for Illumina sequencing using the KAPA Library Preparation Kit (Kapa Biosystems). Fragments ranging from 350–600 bp were selected using the BluePippin (Sage Science) according to the manufacturer’s instructions. Libraries were quantified with the KAPA Library Quantification kit (Kapa Biosystems) and sequencing of the samples was performed on a HiSeq 2500 platform (Illumina) for 301 cycles (150 bp paired ends). Each sample was attributed a total of 2 million paired end reads. Mongoose feces associated gemycircularvirus a, b, c and d yielded 262, 295, 37,644, and 356 reads respectively. The Badger feces associated gemycircularvirus yielded 88 reads and the RNA2 of the Nodavirus 20 reads.
Genomic and phylogenetic analysis
Raw reads were trimmed for quality and adapters using Skewer  and were de novo assembled into contigs using SPAdes . Scaffolds were classified using a tBLASTx search against all complete viral genomes in GenBank using an e-value cut-off of 10−10. Scaffolds with a significant tBLASTx hit were retained and used for a second tBlastx search against the GenBank nucleotide database using an e-value of 10−4 . Open reading frames (ORF) were identified with ORF Finder analysis tools and the conserved motifs in the amino acid sequences were identified with HMMER . Amino acid alignments of the viral sequences were performed with MAFFT version 7  using the --auto option. Maximum likelihood phylogenetic trees were constructed in MEGA6.0 , using JCC (best substitution model) with 500 bootstrap replicates. Potential intron acceptor and donor sites from the novel gemycircularviruses were identified manually. Using the method above we were able to retrieve the five complete gemycircularvirus and partial sequence of the RNA2 of the Lunovirus. Presence of the discovered novel viruses was then confirmed by PCR and Sanger sequencing using the original extracts. Gemycircularviruses’ primers were designed in the capsid gene, covering the complete circular genome and the nodavirus primer pair targeted the RNA2 sequence found. All sequences from the novel viruses were submitted to GenBank [KP263543, KP263544, KP263543-KP263548, KP263546, KP263547, KP263548].
NCN and MZ were supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT Vlaanderen). We would like to thank Prof. Maria São José Nascimento and Joren Raymenants who assisted in the proof-reading of the manuscript.
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