Detection of Diverse Novel Bat Astrovirus Sequences in the Czech Republic

Introduction

Bats (Chiroptera) are important reservoirs of viruses, including zoonotic viruses. Astroviruses (AstVs) in bats were first reported in 2008 from Hong Kong (Chu et al. 2008). Since then, only a few further studies have been carried out that revealed that bats are natural hosts of AstVs in Asia (Zhu et al. 2009, Xiao et al. 2011), North America (Li et al. 2010), and Europe (Drexler et al. 2011, Kemenesi et al. 2014a, b). AstVs seem to be highly prevalent in bats without causing overt clinical disease (De Benedictis et al. 2011). Phylogenetic analyses of partial open reading frame sequences of known AstVs revealed that bat AstVs are not only divergent from previously described human and other animal AstVs, but also show remarkable diversity among themselves (Xiao et al. 2011). On the basis of genetic analysis of the complete capsid region, bat AstVs are classified into genogroup II of the mamastroviruses, together with ovine, mink, and novel human AstVs (Bosh et al. 2011). Widespread infections across many animal species, high genetic diversity, and the possibility of recombination events may favor the emergence of novel, potentially zoonotic AstVs (De Benedictis et al. 2011).

In the present study, we investigated the occurrence and genetic diversity of AstVs in various species of bats in the Czech Republic. We compared the partial genome sequences of detected AstV strains with published sequences and then conducted a phylogenetic analysis.

Materials and Methods

A total of 43 fecal or intestinal content samples from different European bat species were collected in 2008-2014 in South Moravia, Czech Republic (Table S1) (Supplementary Data are available at www.liebertonline/vbz/). Forty intestinal content samples were obtained from bats that died accidentally or from carcasses collected in nature. Three fecal samples were collected from mist-netted individuals during regular trapping and examination of bats by trained chiropterologists. One fecal sample was a mixed sample from two bat species (Pipistrellus nathusii and P. pygmaeus). The bat collection comprised 15 different species from two families. The first family was vesper bats (Vespertilionidae): Eptesicus nilssonii (n=1), E. serotinus (n=1), Hypsugo savii (n=4), Myotis bechsteinii (n=1), M. daubentonii (n=3), M. emarginatus (n=1), M. mystacinus (n=1), Nyctalus noctula (n=7), P. nathusii (n=1), P. pipistrellus (n=12), P. pygmaeus (n=1), Plecotus auritus (n=2), Pl. austriacus (n=2), and Vespertilio murinus (n=5). The second family was horseshoe bats (Rhinolophidae): Rhinolophus hipposideros (n=2). Sampling was performed in compliance with Act No. 114/1992, on Nature and Landscape Protection, and was based on permits 01662/MK/2012S/00775/MK/2012 and 00356/KK/2008/AOPK issued by the Agency for Nature Conservation and Landscape Protection of the Czech Republic.

Total RNA was extracted with the QIAamp Viral RNA Mini Kit (Qiagen) from the supernatant of 10% (vol/vol) fecal or intestinal content suspensions in phosphate-buffered saline (PBS; 0.1 M, pH 7.2) following the manufacturer's protocol. Reverse transcription was carried out in a 20-μL volume containing 150 ng of random hexamers using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany) according to the manufacturer's recommendations. Random hexamer-generated cDNA was screened for the presence of AstV nucleic acid by seminested PCR that targeted the most conserved region of the RNA-dependent RNA polymerase (RdRp) gene (Chu et al. 2008). Resulting amplicons were purified with the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI) and sequenced commercially (SEQme, Dobříš, Czech Republic).

To determine whether the mixed fecal sample from two bat species contained diverse AstV strains, the PCR product was cloned into pCR2.1-TOPO plasmids (Invitrogen, Germany). Multiple clones were picked, purified with a PureLink Quick Plasmid Miniprep Kit (Invitrogen, Germany), and sequenced with M13 forward and reverse primers. The RdRp nucleotide dataset was translated into amino acids and aligned using the ClustalW algorithm in BioEdit 7.0.5.3 with default parameters (Hall 1999). The edited nucleotide dataset was used for phylogenetic analysis. Bayesian inference tree was computed with MrBayes 3.1.2 (Huelsenbeck and Ronquist 2005), with five million generations under the general time reversible (GTR)+I+γ model with gamma distribution in six categories. Consensual topology was created based on 45,000 trees.

Results

Out of 43 samples from 15 different bat species, nine samples (20.9 %) from nine species (E. serotinus, H. savii, M. emarginatus, M. mystacinus, N. noctula, P. nathusii or pygmaeus, P. pipistrellus, V. murinus, and R. hipposideros) tested positive for astrovirus RNA (Table S1). For the mixed sample (P. nathusii and P. pygmaeus), we could not determine which of the two species harbored the virus. Sequencing results of multiple clones of the PCR product confirmed that the mixed sample contained only one AstV strain.

Phylogenetic analysis based on partial (336 nucleotides) RdRp gene sequences clustered the newly described AstV sequences together with other bat AstV strains within the genus Mamastrovirus (Fig. 1). Of the nine AstV sequences detected in this study, seven were selected for phylogenetic analysis. Two sequences were not suitable for phylogeny because of insufficient length. All but one of the AstV sequences grouped (with high posterior probability) together in a single monophyletic lineage, along with the Hungarian sequence BatAstV/BS50/HUN/2013. The sequence detected in R. hipposideros was the most diverse and clustered with two Chinese strains originating from bats of the families Rhinolophidae and Hipposideridae (Zhu et al. 2009) (Fig. 1). The Czech bat AstV sequences showed 68.7–99.3% amino acid identities to one another. The genome sequences of AstV strains described in this study were deposited in GenBank under accession numbers KP843558–KP843564.

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FIG. 1.

Bayesian phylogenetic tree based on a 336-nucleotide fragment within the RNA-dependent RNA polymerase (RdRp) gene of selected astroviruses. Sequence Turkey_AstV/USA (EU143843) was used as the outgroup. Numbers at nodes indicate Bayesian posterior probability. Astrovirus sequences determined in this study are highlighted and in bold.

Discussion

In the present study, we detected AstV sequences in nine out of 15 investigated bat species from South Moravia, and in six of them for the first time: E. serotinus, H. savii, M. mystacinus, P. pipistrellus, V. murinus, and R. hipposideros. Some species (e.g., E. serotinus, M. emarginatus, M. mystacinus, and R. hipposideros) harbored AstV sequences, even though their sample number was small. These findings are consistent with previous studies, which showed that many species of insectivorous bats can carry astroviruses (Chu et al. 2008, Kemenesi et al. 2014b). Interestingly, AstV sequences originating from different bat species of the family Vespertilionidae clustered together and shared higher rates of sequence identities than with AstV sequences derived from the same bat species but from different geographic areas. This observation is supported by a partial result: i.e., the sequence detected in M. mystacinus and sequence identified in the mixed fecal sample from P. nathusii/pygmaeus, shared a high level of amino acid sequence identity (99.3%) and likely represent the same AstV strain circulating in different species of bats. However, the only Czech AstV sequence demonstrated in a bat of the family Rhinolophidae (horseshoe bats) clustered differently; this sequence clustered with AstV sequences of Chinese bats from the same family and a related family. According to our results and previous reports (Zhu et al. 2009, Xiao et al. 2011), there is no significant phylogenetic clustering of bat AstVs among the different bat species and geographical regions. Viruses isolated from a single bat species at a single habitat could be clustered into different groups. On the other hand, viruses detected in different habitats could be clustered within a single group (Xiao et al. 2011).