Identification and characterization of deer astroviruses

Astroviruses (family Astroviridae) are small non-enveloped viruses with a positive-strand RNA genome of between 6.8 and 7.9 kb in length. The ssRNA has a poly(A) tail at the 3′ end, but no 5′ cap. The genome is arranged into three ORFs, with an overlap of approximately 70 nt between ORF1a and ORF1b that encode the protease and RNA-dependent RNA polymerase (RdRP), respectively. The remaining ORF is known as ORF2 and encodes the capsid precursor protein. Astroviruses are 28–35 nm in diameter, icosahedral viruses that have a characteristic five- or sixpointed star-like surface structure when viewed by electron microscopy. Astroviruses (classified as genus Mamastrovirus) have now been identified in several mammalian species, for example cat, swine, sheep, mink, cheetah, sea lion, bottlenose dolphin, dog and bat. Other astroviruses (classified as genus Aviastrovirus) have been identified in avian species such as duck, chicken and turkey (Atkins et al., 2009; Indik et al., 2006; Lukashov & Goudsmit, 2002; Mittelholzer et al., 2003; Rivera et al., 2010; Strain et al., 2008; Toffan et al., 2009; Zhu et al., 2009).

Generally, astrovirus infections are associated with enteric disease with mild to severe signs and symptoms, such as diarrhoea and vomiting (Moser & Schultz-Cherry, 2005). In humans, they cause diarrhoea lasting for 2–4 days, mostly in children <2 years of age, elderly persons or immunocompromised persons (Moser & Schultz-Cherry, 2005). Epidemiological studies suggest that the eight closely related human serotypes 1–8 are responsible for up to 10 % of sporadic, acute, non-bacterial cases of diarrhoea in children (Glass et al., 1996; Klein et al., 2006; Soares et al., 2008) and 0.5–15 % of outbreaks (Akihara et al., 2005; Lyman et al., 2009; Svraka et al., 2007). In some cases, astroviruses can cause extra-enteric manifestations, as is the case for avian nephritis virus that causes interstitial nephritis and growth retardation in young chickens (Imada et al., 2000) and duck astrovirus that causes severe hepatitis (Gough et al., 1984).

At the beginning of 2010, we were notified of an outbreak of gastrointestinal illness in a dense population of European roe deer (Capreolus capreolus) in Denmark. Faecal materials from ten animals with diarrhoea were analysed for the presence of bacteria, parasites and bovine viral diarrhea virus and were consistently found to be negative. It has been shown previously that sequence-independent amplification of known and unknown RNA viruses in clinical diarrhoea samples was an efficient procedure for virus identification (Allander et al., 2001, 2005; van Leeuwen et al., 2010). Therefore, the ten faecal samples from roe deer were collected for the present study, anonymized, annotated with VS numbers, and stored at −80 °C until the analysis for the presence of RNA viruses using sequence-independent amplification was carried out.

Large-scale molecular RNA virus screening, based on host nucleic acid depletion, sequence-independent amplification and sequencing of partially purified viral RNA, was performed on two pools of nucleic acid isolated from five diarrhoea samples each, as described previously (van Leeuwen et al., 2010). Diarrhoea samples were suspended in PBS to 10 % weight-to-volume and centrifuged at 14 000 r.p.m. (Eppendorf microcentrifuge) for 5 min. Supernatants were filtered through 0.45 μm spin filters (Ultrafree-MC; Millipore) and treated with Omnicleave Endonuclease (Epicentre Biotechnologies). Viral RNA was extracted with the Nucleospin RNA XS kit (Machery-Nagel) and cDNA was generated using Superscript III reverse transcriptase (Invitrogen) and primer FR26RV-N (5′-GCCGGAGCTCTGCAGATATCNNNNNN-3′). After second strand synthesis using 3′–5′exo⁻ Klenow DNA polymerase (New England Biolabs) and creating pools of nucleic acid from five diarrhoea samples per pool, PCR amplification was performed with primer FR20RV (5′-GCCGGAGCTCTGCAGATATC-3′) and AmpliTaq Gold (Applied Biosystems). PCR products were separated on an agarose gel and fragments were excised, purified using the Invisorb Spin DNA Extraction kit (Invitek GmbH) and cloned into pCR4-TOPO (Invitrogen). Sequencing templates were produced directly from colonies in a 96-well format by performing a colony PCR with M13 primers and Taq DNA polymerase (New England Biolabs). Sequencing was performed on purified PCR products using Big Dye Terminator V3.1 Cycle Sequencing kit (Applied Biosystems) and a 3130XL or 3700 genetic analyser (Applied Biosystems). A total of 190 clones were analysed according to nucleotide and translated nucleotide blast searches (Altschul et al., 1997). Most of the sequences in each pool were unclassified or of bacterial origin. One clone in the first pool containing samples VS3500001–VS3500005 revealed the presence of an astrovirus by using the translated nucleotide blast search (Fig. 1⇓).

To determine whether astroviruses are present in the deer faecal samples, a degenerate RT-PCR assay using primers that target conserved motifs in the RdRP gene of diverse astroviruses (Kapoor et al., 2009) was used (Fig. 1⇑). Two of the ten faecal samples, VS3600005 and VS3600006, were positive for an astrovirus that was distinct from the previously identified astroviruses upon sequencing and calculation of genetic distances (p-distance) between different astrovirus species (Table 1⇓). The astrovirus fragment identified using large-scale molecular virus screening was therefore derived from sample VS3600005. Although it proved difficult to acquire more genome sequences, probably due to the nature of the sample and low virus titres, the 3′ ∼3000 bp from the deer astrovirus genomes could be amplified using 3′RACE PCR, using primer VS430 (5′-GAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTT-3′) and Superscript III reverse transcriptase (Invitrogen) in first strand cDNA synthesis (van Leeuwen et al., 2010). Oligonucleotides VS431 (5′-GCGAGCACAGAATTAATACGACT-3′) and VS544 (5′-GGGAAGGATATTGAGACTCTC-3′) were used in a PCR amplification, using the Expand Long Template PCR system (Roche), according to instructions of the manufacturers (Fig. 1⇑). All sequences were determined on at least two clones in both directions and submitted to GenBank (accession nos HM447045 and HM447046) . Amino acid sequences from ORF1b and ORF2 of the astroviruses from samples VS3600005 and VS3600006 were aligned to the respective partial and complete ORFs of other astroviruses in GenBank using clustal x2 (Larkin et al., 2007). Neighbour-joining phylogenetic trees were generated using amino acid p-distances implemented by using the program mega4 (Fig. 2⇓) (Tamura et al., 2007). This analysis demonstrated that the astroviruses from samples VS3600005 and VS3600006, which we named CcAstV-1 and CcAstV-2, respectively, were most closely related to each other and constituted a separate phylogenetic clade, indicating that they are a novel species within the genus Mamastrovirus.

This report documents the first identification of astroviruses in roe deer. Within the genus Mamastrovirus two large phylogenetic clades seem to exist, with the first group consisting of the human VA1–3, mink, sheep, bat and sea lion-1 astroviruses and the second group which contains the human 1–8, MLB1, MLB2, cat, swine, dog, bottlenose dolphin, sea lion-2 and -3 astroviruses, and also the novel astrovirus species from roe deer. The phylogenetic topology determined in this study is largely in agreement with previous analyses of astrovirus phylogeny (Finkbeiner et al., 2008, 2009a, b, c; Kapoor et al., 2009; Rivera et al., 2010). The genetic distances between the roe deer astroviruses and other astroviruses were generally greater in the capsid region than in the RdRP region. This is consistent with other studies and is generally thought to be the result of strong positive selective pressure on the capsid-coding region from the host immune system (van Hemert et al., 2007). As the genetic distance between the two roe deer astroviruses is at least as large as between the eight human astrovirus serotypes 1–8, CcAstV-1 and CcAstV-2 may constitute two different serotypes. Whether CcAstV-1 and CcAstV-2 were responsible for causing diarrhoea in the roe deer remains to be determined.

As (re)emerging infectious diseases pose a continuous health threat to wild and domestic animals as well as to humans, more attention should be paid to combat newly emerging viral diseases and epidemics. One approach is to obtain a thorough understanding of the diversity of viruses in wildlife, which provides epidemiological baseline information about pathogens and may lead to early identification of newly emerging pathogens in humans in the future (Kuiken et al., 2005). The discovery of two novel astroviruses from wild deer described here is an example of the needed expansion of our knowledge of the virus diversity present in the wildlife reservoir. Sequence-independent amplification of viral nucleic acids, which we used to discover these viruses, provides a relatively simple, unselective technology with diagnostic capabilities to identify novel viral species.