key: cord-0935356-cmx3q2sc authors: Amoroso, Maria Grazia; Russo, Danilo; Lanave, Gianvito; Cistrone, Luca; Pratelli, Annamaria; Martella, Vito; Galiero, Giorgio; Decaro, Nicola; Fusco, Giovanna title: Detection and phylogenetic characterization of astroviruses in insectivorous bats from Central‐Southern Italy date: 2018-06-12 journal: Zoonoses Public Health DOI: 10.1111/zph.12484 sha: dab1297089706b1f2ce25af936868f08d0d2ecc8 doc_id: 935356 cord_uid: cmx3q2sc In recent years, bats have been found to harbour many viruses, raising several questions about their role as reservoirs and potential disseminators of zoonotic viruses. We investigated the presence of six virus families in bats in three regions of Central‐Southern Italy. Astroviruses were identified in seven of 13 bat species. Sequence analysis revealed marked genetic heterogeneity among the astroviruses identified, with nucleotide identity ranging between 60.26% and 87.62%. Astrovirus diversity was not associated with the bat species, the geographic areas or the bat colony, suggesting the circulation of several astrovirus strains in Italian ecosystems. Genetic diversification and interspecies transmission appear common in bat astroviruses and could provide, potentially, the bases for transmission to humans and other mammals. Yet overemphasizing this risk might have detrimental consequences for bat conservation and preservation of the important ecosystem services bats provide. risk is crucial in Western countries. Several bat species avoid human settlements and have little chance to enter into contact with humans (Russo & Ancillotto, 2015) , and other species are at risk of extinction (e.g. Conenna, Rocha, Russo, & Cabeza, 2016; O'Shea, Cryan, Hayman, Plowright, & Streicker, 2016) , making the risks negligible. However, increasing anthropization of the environment is altering the ecosystems, disrupting usual habitats and/or creating novel ecological niches that may overlap with human activities. It should be considered, however, that bats provide important ecosystem services (Aizpurua et al., 2018; Ancillotto et al., 2017; Boyles, Cryan, McCracken, & Kunz, 2011; Kunz, Braun de Torrez, Bauer, Lobova, & Fleming, 2011) . Overemphasizing the potential risks posed by bats to humans may generate unjustified alarmism, a fact that has raised considerable concern in conservationists (López-Baucells, Rocha, & Fernández-Llamazares, 2017) , as also highlighted by the EUROBATS agreement on the conservation of bat populations in Europe (working group on "Communication, Bat Conservation and Public Health"). Under this perspective, surveillance may still be important to avoid or mitigate potential conflicts and eventually improve bat conservation policies. In Italy, a few studies have been carried out to assess the presence of viruses in bats Lelli, Papetti, et al., 2013) but only in the Northern regions. These studies detected orthoreoviruses and coronaviruses in various bat species. However, there are no data for the Central-Southern Italian regions, which are characterized by a warmer, drier climate and a different biogeography. In this study, we screened 13 bat species living in these geographic areas. The bat species were selected ad hoc to represent a range of environmental and behavioural differences. Our data set covered species that form large colonies in caves and show high fidelity to their roosting sites (e.g. Miniopterus schreibersii); species that roost in trees in small groups and switch roosts frequently (e.g. Barbastella barbastellus); and synanthropic species that are more likely to enter into contact with humans (e.g. Pipistrellus kuhlii). The bats were screened for a large panel of viruses, including rabies viruses, coronaviruses, reoviruses, caliciviruses, astroviruses and enteroviruses. We overall sampled 147 individuals from three regions of Central-Southern Italy. Most bats we screened were caught on emergence from their roosts with harp traps or at drinking sites with mist nets. In such cases, bats were promptly removed from the trap or the net and their forearm length and body mass were measured, respectively, with a caliper to the nearest 1 mm and a digital scale to the nearest 0.1 g. Sex was assessed by inspecting genitalia (Racey, 1988) , and wings were trans-illuminated to distinguish juveniles from adults (Anthony, 1988) . In females, we ascertained pregnancy by palpation (Racey, 1988) , and lactation by the presence of enlarged nipples surrounded by a hairless skin area and by extruding milk with a gentle finger pressure on the nipple base. Bats were immediately released after processing. A few samples came from animals hosted at rehabilitation centres. Saliva was sampled from each bat with a dry sterile swab, which was placed in viral transport medium (Chu, Poon, Guan, & Peiris, 2008) , and transported in a cool box to the laboratory for the analysis. Bat droppings expelled during manipulation of specimens were collected, stored in sterile vials and preserved in cool boxes during transportation. Nucleic acids were extracted with the MagMax automated system The presence of rabies virus and MERS-coronavirus (MERS-CoV) was investigated by real-time RT-PCR using two commercial kits, that is Rabies virus Real Time RT-PCR kit (Shanghai ZJ Bio-Tech Co., Ltd) and MERS-CoV Real Time RT-PCR kit (Shanghai ZJ Bio-Tech Co., • Identification of astroviruses in four bat species never found positive before. • Identification of astrovirus in bats living in Italy. • Strains of astrovirus identified revealed a high degree of genetic diversity, not related to bat species. • Mechanisms other than host specificity may drive virus genetic diversification. Ltd). The reaction was carried out following the manufacturer's instructions. All the tests were performed with a 7500 Fast Real time PCR system (Applied Biosystems, Monza, Italy). Positive and negative controls were included in the kits. The primers employed for detection of the different viruses are indicated in Table 1 . All the assays were carried out with the Flexid Mastercycler NexsusX2 (Eppendorf) using the SuperScript™One- Step RT-PCR kit (Life Technologies Italia). Coronaviruses (CoVs) were searched as described by Drosten et al. (2003) (Table 1) . For reovirus detection, the nucleic acids were preventively denatured (2 μl viral extract with 1.4 μl of DMSO at 97°C for 5 min) and then reversetranscribed and amplified using a nested protocol, as described previously (Leary et al., 2002; All the PCR products were analysed by Tape Station 2200, an automated platform for electrophoresis, (Agilent Technologies), using the D1000 screentape system. Amplicons of the PCR-positive samples were sequenced as previously described (Amoroso et al., 2013) . The nucleotide sequence similarity searches were performed using the BLAST server (http:// www.ncbi.nlm.nih.gov/genbank/index.html). Nucleotide sequences of AstVs were aligned using the program Clustal W (Larkin et al., 2007) with reference sequences of Mamastrovirus (MAstV), using an avian astrovirus (AvAstV) JF414802 as outgroup (Supporting Information Table S1 ). Phylogenetic analyses were carried out by Mr Bayes (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003) program implemented in the software package Geneious v. 9.1.8 (Biomatters, New Zealand). Bayesian inference was performed using four "chains" run over one million generations (with the first 2000 trees discarded as "burn-in") and supplying statistical support with subsampling over 200 replicates. jModelTest (Posada, 2008) All the screened bats were negative to reoviruses, caliciviruses, enteroviruses, rabies viruses and MERS-CoV. When analysed for the Bat species B. barbastellus (7.14%) and in an additional four bat species (Table 2) . Geographic location of sampling sites from which positive samples were taken is indicated in Figure 1 . Upon interrogation (November 2017) of GenBank sequence database ( (Table 3) . TA B L E 3 Interrogation by BLAST search of NCBI nucleotide database (December 2017) based on the ORF1b (RdRp) sequences generated in this study. The strain with the highest % nucleotide identity (% PI) and E value (E-v) found in the database (BM, best match) is shown for each bat sequence generated in this study (Table 3) . Upon sequence comparison, a marked genetic heterogeneity was Information Table S1 ) and supported by bootstrap values ≥95. In this study, we investigated the presence of various human viral pathogens in 14 different species of bats captured in Central and Southern Italy. None of the samples was positive to rabies virus that is the most serious concern for the potential human transmission. From an epidemiologic point of view, bat-associated rabies cases are rare, with the incidence rates in Canada and the Unites States being as low as 2.2-6.7 human cases per billion persons/year over a 57-year period (Velasco-Villa et al., 2017) . However, there is still a potential risk that needs attention, especially for bat specialists and rehabilitators, who handle bats and are often exposed to bites. Our study, in spite of the relatively small number of sampled animals, confirms that this risk is negligible in Italian territories. Consensus diagnostic molecular assays are useful to detect novel viral species or genetically different viral strains, but they are usually not highly sensitive. Using broadly reactive consensus primers, CoV RNA was detected in 10/147 (6.8%) samples. These results could not be confirmed with sequence analysis. However, the samples also tested negative by a quantitative assay specific for MERS-CoV, thus ruling out the presence of this virus in the sampled population. Using consensus primers universal for the Astroviridae family, in Europe and Asia (Drexler et al., 2011; Fischer et al., 2016 Fischer et al., , 2017 Kemenesi et al., 2014; Zhu et al., 2009) . The RT-PCR prevalence (22.45%) observed in our study was higher than that observed in Hungary (6.94%) (Kemenesi et al., 2014) and similar to that found in Germany (25.8%; Fischer et al., 2016) . AstV in 46% of the tested bats (Chu et al., 2008; Xiao et al., 2011) . The prevalence was also found to greatly vary by bat species (Fischer et al., 2017) . As an example, a study carried out in China revealed the presence of Astrovirus in 83.3% of the samples of Myotis pylosus analysed, whilst the prevalence was found much lower for other species (Chu et al., 2008) . To detect and characterize the AstV strains, we amplified and sequenced a fragment of 422 bp of the RdRp gene, which represents the most conserved region of the AstV genome. Upon sequence analysis, we observed a remarkable genetic diversity among the various bat AstV strains detected in Italy. Such a high degree of variation was observed also within species and in the same geographic area or colony. These findings do not confirm the bat species specificity of AstV, proposed by other authors (Fischer et al., 2016 Astrovirus infection is associated with gastro-enteritis in most animal species, and humans AstVs are regarded as a common cause of viral diarrhoea in children (Mendez, Aguirre-Crespo, Zavala, & Arias, 2007; Xiao et al., 2017) . Avian AstVs have also been associated with extra-intestinal diseases, such as nephritis in chicken (Imada et al., 2000) and hepatitis in ducks (Todd et al., 2009) . Recently, AstVs have been also detected in the nervous tissues of minks with shaking disease (Blomstrom, Widen, Hammer, Belak, & Berg, 2010) and of bovines with neurological disorders (Bouzalas et al., 2014; Li et al., 2013) . Neurological disease in immunocompromised human patients has been associated with AstV infection (Brown et al., 2015; Fremond et al., 2015; Lum et al., 2016; Quan et al., 2010) . In our research, all the animals positive to AstV appeared healthy, as also reported in previous studies in bats (Fischer et al., 2017) . Astroviruses could therefore simply be nonpathogenic members of the bat virome. However, more information is needed on bat-borne immune response to state whether these viruses are really nonpathogenic for bats. Bats could play an important role in transmitting such viruses to humans, as AstV transmission usually follows an oral-faecal route. Contamination of food or drinking water could for example occur by bat droppings. On this regard, it is however important to underline that the probability that human ingest food and water contaminated by AstV coming from human faeces looks much higher-see, for instance, the high percentage (28.70%) of human AstVs recently found in mussels harvested in the Gulf of Naples, Italy (Fusco et al., 2017) . Interspecies transmission of AstV has been documented on more occasions (De Battisti et al., 2012; De Benedictis, Schultz-Cherry, Burnham, & Cattoli, 2011; Mihalov-Kovacs et al., 2017; Nagai et al., 2015) . Also, novel human AstVs (MLB1, MLB2, VA1, HMO-C, HMO-B, HMO-A, VA-2) have been identified that are genetically unrelated to "classical" human AstVs (Banyai, Meleg, Moschidou, & Martella, 2010; Finkbeiner, Le, Holtz, Storch, & Wang, 2009; Finkbeiner, Li, et al., 2009; Kapoor et al., 2009 ) and closer to animal AstVs. The origin of animal-like human AstVs has not been deciphered yet. The potential zoonotic risks associated with bats have attracted the attention of researchers, mostly after the discovery of SARS-like and MERS-like CoVs (two coronaviruses highly pathogenic for humans) in European bat species, although the zoonotic risks posed by bat viruses, likely very limited, should be assessed more properly (Kohl & Kurth, 2014) , in large structured studies. Thanks go to Luciano Bosso for helping with the preparation of Figure 1 . 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