Archives of Virology (2019) 164:3065–3071 https://doi.org/10.1007/s00705-019-04414-7 BRIEF REPORT Analysis of a novel RNA virus in a wild northern white‑breasted hedgehog (Erinaceus roumanicus) Gábor Reuter1 · Éva Várallyay2 · Dániel Baráth2 · Gábor Földvári3,4 · Sándor Szekeres4 · Ákos Boros1 · Beatrix Kapusinszky5 · Eric Delwart5,6 · Péter Pankovics1 Received: 11 June 2019 / Accepted: 23 August 2019 / Published online: 23 September 2019 © The Author(s) 2019 Abstract Tombusviruses are generally considered plant viruses. A novel tombus-/carmotetravirus-like RNA virus was identifed in a faecal sample and blood and muscle tissues from a wild northern white-breasted hedgehog (Erinaceus roumanicus). The complete genome of the virus, called H14-hedgehog/2015/HUN (GenBank accession number MN044446), is 4,118 nucleo- tides in length with a readthrough stop codon of type/group 1 in ORF1 and lacks a poly(A) tract at the 3′ end. The predicted ORF1-RT (RdRp) and the capsid proteins had low (31-33%) amino acid sequence identity to unclassifed tombus-/noda-like viruses (Hubei tombus-like virus 12 and Beihai noda-like virus 10), respectively, discovered recently in invertebrate animals. An in vivo experimental plant inoculation study showed that an in vitro-transcribed H14-hedgehog/2015/HUN viral RNA did not replicate in Nicotiana benthamiana, Chenopodium quinoa, or Chenopodium murale, the most susceptible hosts for plant-origin tombusviruses. Recently, the discovery of novel viruses has been dramati- viruses are traditionally thought to have a narrow host range cally enhanced by the use of culture-free viral metagenomics but might actually have a much wider host range [2]. and next-generation sequencing techniques [1]. However, in For example, a recent study investigating the RNA virome many cases, this approach does not allow the host species of invertebrate animals identifed 1,445 novel viruses by of the novel virus to be identifed. In addition to the host viral metagenomics, including many tombus- and tombus- origin, dietary sources or other forms of contamination are like viruses [3]. Tombusviruses (family Tombusviridae) also possible sources of certain viruses, especially in faecal are positive-sense, single-stranded RNA viruses (genome specimens. The picture is complicated by the fact that some length, 3.7 to 4.8 kb) that are currently thought to be plant viruses with relatively narrow host range [4]. The origin and pathogenic roles (if any) of tombus-like viruses in inverte- brate specimens are presently uncertain. On the other hand, Handling Editor: Massimo Turina. a relative of the tombusviruses, Providence virus (the only Carmotetraviridae * Gábor Reuter member of the family ), a positive-sense, [email protected] single-stranded RNA virus, is thought to be an insect virus [5, 6] that replicates in lepidopteran species (Helicoverpa 1 Department of Medical Microbiology and Immunology, zea), although a recent study indicated that it was also able Medical Center, University of Pécs, Szigeti út 12, Pecs 7624, Hungary to replicate in the human HeLa cell line [7, 8]. These data show that the possible full spectrum of host and target cells 2 Agricultural Biotechnology Institute, National Agricultural Research and Innovation Centre, Gödöllő, Hungary infected by members of some viral families may be wider 3 than currently appreciated. Evolutionary Systems Research Group MTA Centre Erinaceus rou- for Ecological Research, Tihany, Hungary The northern white-breasted hedgehog ( 4 manicus) is a medium-sized mammal belonging to the genus Department of Parasitology and Zoology, University Erinaceus, of Veterinary Medicine, Budapest, Hungary family Erinaceidae (Mammalia: Eulipotyphla). It can be found in Central Europe, the Balkan Peninsula, 5 Vitalant Research Institute, San Francisco, CA, USA the Adriatic Islands, Ukraine and Russia, extending to the 6 University of California, San Francisco, San Francisco, CA, Ob River in Siberia. These animals inhabit farmland, parks USA Vol.:(0123456789)1 3 3066 G. Reuter et al. and gardens in rural and urban areas, scrubby habitats at contig to identify the specifc viral RNA from the specimen the edge of forests, and shrubby vegetation [9]. They feed pool. Diferent sets of specifc primers were designed based on earthworms, insects (larvae, pupae and imagos), snails, on the metagenomic reads and contigs to obtain the com- slugs, small vertebrates (amphibians, lizards and occasion- plete viral genome sequence (H14-hedgehog/2015/HUN) ally young rodents), chicks, bird eggs, and even some berries and for verifcation of the full-length metagenomic contigs and fruits. by the primer-walking [13] and 5′/3′RACE methods [14]. Here, we report the complete genome sequence of a Primer sequences are available upon request. PCR products novel positive-sense, single-stranded RNA virus from a were sequenced directly using an automated sequencer (ABI wild northern white-breasted hedgehog; however, the host PRISM 310 Genetic Analyzer, Applied Biosystems, Staf- origin of the studied virus could not be determined in spite ford, USA). All faecal specimens (N = 20) from northern of in vivo experimental inoculation studies with the most white-breasted hedgehogs were tested by RT-PCR using the susceptible plants for tombusviruses. SCR-F/R primers. In addition, available coagulated blood, A total of 20 faecal specimens were collected from ear skin, abdominal muscle, spleen and liver tissues col- northern white-breasted hedgehogs [10]. Eight faecal sam- lected from animal H14 were also tested by RT-PCR using ples (H1, H2, H4, H9-H11, H13 and H14) were collected SCR-F/R primers. between April and June in 2015 from carcasses of road- Coding regions were identifed using NCBI Open Read- killed hedgehogs from urban environments, seven of which ing Frame Finder (https://www.ncbi.nlm.nih.gov/orf nder/ ). were from streets in the capital, Budapest, and one was from All evolutionary analyses were conducted in MEGA 6.06 the suburb of Szentendre, Hungary. Twelve faecal samples [15]. The non-structural (RNA-dependent RNA polymerase) (MR1-MR9 and MR11-MR13) were collected from wild- amino acid (aa) sequences of the study strain and the repre- living hedgehogs collected from April to July in 2015, from sentative reference viruses were aligned using MEGA 6.06 a natural wild area near the town of Pécs, Baranya County, and pre-tested using a best amino acid model (ML) search. Southwest Hungary. H10 and H11 were juvenile animals Dendrograms were constructed by the maximum-likelihood (< 1 year old), and the others were adults (> 1 year old), method based on the Poisson model with gamma distribution as judged based on their size and – when available – skull (+G) and invariable sites (+I). To assess the reliability of the bone ossifcation. Samples were collected by qualifed biol- trees, 1000 replicates of bootstrap analysis were performed. ogists with valid permission from the National Inspector- The genomic DNA of H14-hedgehog/2015/HUN was ate for Environment, Nature and Water (4018-4/2015). A cloned for in vitro transcription and in vivo plant inocu- specimen pool containing three faecal samples (H9, H13 lation studies. Briefly, primers were designed based on and H14) was randomly selected for viral metagenomics the sequence of the putative H14-hedgehog/2015/HUN analysis [10]. Briefy, the specimen was diluted in PBS, (H14_HedgehogT7_1F, TAA TAC GAC TCA CTATA passed through a 0.45-μm sterile flter (Millipore), and cen- GGG TAC ATT ATG GCC CAA AAT GAT G; H14_ trifuged at 6,000 × g for 5 min. The fltrate was then treated Hedgehog_4118RXbaI, TCT CTA GAC TCG AGCA CTT with a mixture of DNases and RNases to digest unprotected ATC TTG TAC TAG GTT ATC ; bold letters represent nucleic acids [11]. Viral-particle-protected nucleic acids sequences from the virus under study). cDNA was synthe- were extracted using the QIAamp spin-column technique sized using a RevertAid First Strand cDNA Kit (Thermo (QIAGEN) and subjected to a viral metagenomic analysis Fisher Scientifc) according to the manufacturer’s instruc- using sequence-independent random amplifcation [12]. A tions from RNA extracted from hedgehog faeces using the viral cDNA library was constructed using a Nextera XT primer H14_Hedgehog_4118RXbaI. The virus-specific DNA Library Preparation Kit (Illumina) sequenced on a cDNA was then used as a template for PCR amplifcation HiSeq Illumina platform according to the manufacturer’s with Q5 High-Fidelity DNA Polymerase (New England instructions as described previously [11]. The reads were Biolabs) (initial denaturation for 30 s at 98 °C, 40 cycles of then trimmed; assembled de novo, and analyzed using denaturation for 10 s at 98 °C, annealing for 30 s at 70 °C, an in-house pipeline [11]. The reads and contigs greater extension for 3 min at 72 °C, and a fnal extension for 2 min than 100 bp were compared to the GenBank protein data- at 72 °C) using the primers H14_HedgehogT7_1F and base (BLASTx). Virus-family-level categorization of viral H14_Hedgehog_4118RXbaI. The PCR-product was iso- metagenomic reads was based on the best BLASTx-scores lated from a 1.2% agarose gel using a GeneJet Gel Extrac- (E-value ≤ 10−10). Sequence-specifc screening primer pairs tion Kit (Thermo Fisher Scientifc), and it was then ligated (SCR-F, 5′-AAT TCA ATT ACC TAC TGG CCT CAT -3′, cor- into pJET1.2/blunt Cloning Vector (CloneJET PCR Clon- responding to nt positions 2169-2192 and SCR-R: 5′, TCC ing Kit, Thermo Fisher Scientifc), introduced into E. coli TAG CGC GGT GTT CAT GTC TCC -3′, corresponding to nt (DH5alpha) by transformation. The plasmid DNA was puri- positions 2536-2513 of the study strain) were designed based fed from an overnight bacterial culture using a Nucleospin on the RNA-dependent RNA polymerase (RdRp) sequence Plasmid Purifcation Kit (Macherey Nagel). Using restriction 1 3 Novel RNA virus in a hedgehog 3067 endonuclease sites for NotI (site on the pJET1.2 vector) and 23,004,466 reads, 315,105 sequence reads were obtained XbaI, the entire virus genome was excised from pJET1.2 showing translated protein similarity to viral sequences from and then cloned into a pUC18 vector digested with SmaI this sample pool.