key: cord-0722797-j5297kn3 authors: Martella, Vito; Catella, Cristiana; Capozza, Paolo; Diakoudi, Georgia; Camero, Michele; Lanave, Gianvito; Galante, Domenico; Cafiero, Maria Assunta; Lavazza, Antonio; Bányai, Krisztián; Buonavoglia, Canio title: Identification of astroviruses in bovine and buffalo calves with enteritis date: 2020-08-31 journal: Research in Veterinary Science DOI: 10.1016/j.rvsc.2020.04.010 sha: fc55dddbc03e3407909050e21acf7fc3efee67f5 doc_id: 722797 cord_uid: j5297kn3 Abstract Astroviruses (AstVs) have been identified in the stools of calves with enteritis and in the brain tissues of bovines with encephalitis but their pathogenic role has not been clarified. In this study, we report the detection and characterization of bovine and water buffalo AstV strains identified in young bovine and buffalo calves with enteritis in Italy between 2012 and 2015. By negative staining transmission electron microscopy (TEM) observation, AstV-like particles were identified in the stools of the animals and AstV RNA was confirmed molecularly. The sequence (~3.2-kb) at the 3′ end of the genome was determined for two bovine and two buffalo AstVs. Sequence and phylogenetic analysis on the partial ORF1b and full-length ORF2 revealed a marked genetic diversity although the viruses were distantly related to other AstV identified from ruminants. Gathering sequence information on ruminant AstVs is important to understand the extent of inter-species circulation and for the development of reliable, specific diagnostic tools. Astroviruses (AstVs) are a group of small, non-enveloped RNA viruses with an icosahedral capsid of 27-30 nm in diameter (Matsui and Greenberg, 1996) with a peculiar five-or six-pointed star shaped appearance when observed by negative staining electron microscopy (Caul and Appleton, 1982) . The family Astroviridae has been classified by International Committee on Taxonomy of Viruses (ICTV) 1 into two genera, namely Mamastrovirus and Avastrovirus known to infect mammalian and avian species, respectively. The genome length is 6.8 to 7.9 kb and includes a 5′ untranslated region (UTR), followed by three open reading frames (ORFs) namely ORF1a, ORF1b and ORF2, a 3' UTR and a poly-A tail. There is a frame shift between ORF1a and ORF1b. ORF1a and ORF1b encode nonstructural proteins, a serine protease, and an RNA-dependent RNA polymerase (RdRp). ORF2 is expressed from a subgenomic RNA and encodes the viral capsid protein (Mendez and Arias, 2007) . Since the first description of human AstV in children with diarrhea in 1975 (Appleton and Higgins, 1975) , a wide variety of AstVs have been reported in multiple animals including cattle, pigs, sheep, minks, dogs, cats, mice, sea lions, bats, whales, chickens, and turkeys (De Benedictis et al., 2011) . Common clinical signs caused by enteric AstV infection in humans include vomiting and diarrhea. These signs range from mild to severe and affect primarily children and immunocompromised individuals (Moser and Schultz-Cherry, 2005) . Bovine AstV was first described in England in 1978 from acute enteritis in calves (Woode and Bridger, 1978) . The bovine AstV strain UK was initially considered to be avirulent, as experimental infection of two gnotobiotic calves with AstV-like virus did not cause diarrhea (Woode and Bridger, 1978) . In 1984, two bovine AstV isolates from the USA, US1 and US2, antigenically related to the isolate obtained in England (UK), were shown to cause infection and cytopathology of M cells of the dome epithelium covering the Peyer's patches of the calf ileum (Woode et al., 1985) . In addition, co-infection with other enteric pathogens, such as bovine rotavirus (RV) and bovine torovirus, was shown to increase the severity of AstV infection (Woode et al., 1984) . Antigenic differences have been observed among bovine AstV strains, suggesting that multiple serotypes of bovine AstV may exist in nature (Woode et al., 1985) . Even though bovine AstV was one of the earliest mammalian AstV to be discovered and studied in detail, no further research has been performed on this candidate bovine enteric pathogen over the past three decades. The genomic sequence of bovine AstVs was determined only in 2011 (Tse et al., 2011) . Recently, bovine and ovine AstVs have been associated with T encephalitis in cattle and sheep (Li et al., 2013; Bouzalas et al., 2014; Schlottau et al., 2016; Boujon et al., 2017 Boujon et al., , 2019 Pfaff et al., 2017; Seuberlich et al., 2016) , noting similar findings observed in humans (Quan et al., 2010; Vu et al., 2016) and in minks (Blomström et al., 2010) . Altogether, these findings suggest that these enteric viruses may acquire neurotropism, although the mechanisms of this change in tissue tropism are not yet known (Selimovic-Hamza et al., 2017) . In spite of the new perspectives and accumulating evidence on the biological plasticity of AstVs, there is still limited information on the epidemiology and genetic diversity of these viruses in ruminants (Tse et al., 2011; Oem and An, 2014; Alfred et al., 2015; Sharp et al., 2015) , thus hindering to depict a complete portrait of their impact on large ruminants in terms of health and production. This gap may be an obstacle to the development of effective, specific diagnostic tools and unnecessarily delay the development/adoption of measures of prophylaxis. In this study, we report the characterization of bovine and buffalo AstV strains identified from different enteritis outbreaks occurred in Italy in 2012, 2013 and 2015. Between 2012 and 2015 a total of 27 fecal samples from 8 herds located in Southern Italy were submitted to our laboratories. The samples were collected from sporadic cases and small outbreaks of enteritis occurred in six bovine herds, located in Lecce (nr. 1), Potenza (nr. 4) and Foggia (nr. 1) and in two buffalo herds located in Foggia. The animals, aged 1-4 months, displayed severe enteric signs with profuse watery brown to yellow feces and mucus. The signs tended to disappear 5-7 days after their onset in the recovering calves. In a buffalo herd (Foggia 32,013) along with the with acute enteric disease, in some animals the enteric signs tended to configure as a chronic disease, with the calves showing marked delay in growth and hypotrichosis. Morbidity in calves ranged from 5.7% (2/35) to 45% (18/40) ( Table 1) . Mortality ranged from 2.8% (1/35) to 22.5% (9/40). All the samples were stored at −80°C until use. By negative staining TEM microscopy, AstV-like particles were identified in four homogenates of pooled enteric specimens from the four different herds (two from buffalo and two from bovine herds), on the basis of their typical morphologic characteristics (Fig. 1 ). RNA extracts were prepared from 10% fecal homogenates in phosphate-buffered saline (PBS), pH 7.3, after clarification by centrifugation at 10,000 ×g for 3 min. Two-hundred microliters of the supernatants were used for RNA and DNA extraction with the QIAamp cador Pathogen Mini Kit (Qiagen GmbH, Hilden, Germany), following the manufacturer's protocol. Screening for AstV was accomplished by One Step Reverse Transcription (RT)-PCR and nested PCR assays using a broadly reactive set of primers targeting the ORF1b region of AstV (Table 2 ). The specificity of the amplification was confirmed by direct sequencing of the PCR products after gel-excision and elution. Analyses of the sequences with web-based tools (BLAST 2 ) and FASTA 3 with default values were used to find homologous hits. The enteric samples were also screened molecularly for other bovine enteric pathogens, including RV type A (RVA), coronavirus (CoV), calicivirus, pestivirus and Cryptosporidium parvum (Table 2) . Bacteriological examination of the fecal samples and presumptive identification was performed by using conventional standard culture methods with commercially available media (Oxoid, Milan, Italy). Bacterial identifications were achieved by evaluating the biochemical characteristics with the API system (bioMerieux, Marcy l'Etoile, France). The 3′ end of the genome (̴ 3.2 kb) of AstVs was amplified with a 3′-RACE protocol (Scotto-Lavino et al., 2006) using the AstV RdRp universal forward primers described by Chu et al. (2008) and the reverse primer QT (Table 2 ). One step RT-PCR and PCR assays were performed using SuperScript® III First-Strand Synthesis SuperMix (Invitrogen Carlsbad, United States) and LaTakara PCR kit version 2.1 (TaKaRa Bio Europe S.A.S, Saint-Germain-en-Laye, France), respectively. The PCR products were purified using TOPOXL Gel Purification kit (Invitrogen, Carlsbad, United States), cloned into the PCR XL-TOPO vector (Invitrogen, Carlsbad, United States) and sequenced by Eurofins Genomics laboratories (Milano, Italy). The genome annotation was performed by using FindORFs software in Geneious version 9.1.8. The AstV sequences have been submitted in GenBank with accession numbers KT963069-71 and MN718860. Deduced aminoacidic (aa) sequences of partial ORF1b and complete ORF2 the AstVs detected in the study were aligned with cognate sequences of Mamastroviruses (MAstVs) and the Avastrovirus (AvAstV) strain GA2011 (GenBank accession no. JF414802), retrieved from GenBank, using the plugin MAFFT version 1.3.6 (Katoh, 2002) implemented in the software Geneious v. 9.1.8 (Biomatters, New Zealand). The appropriate substitution model settings for the phylogenetic analysis and estimation of selection pressure on coding sequences were derived using jModelTest, based on the least Bayesian Information Criterion (BIC) scores (Posada, 2009) . Phylogenetic analyses were conducted using Mr. Bayes plugin (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003) implemented in the software Geneious version 9.1.8 (Biomatters, New Zealand). P-distance (p-dist), defined as the proportion (p) of aa sites at which the two sequences to be compared are different, was obtained by dividing the number of aa differences by the total number of sites. The p-dist was calculated using the software MEGAX (Kumar et al., 2018) . A total of 4 herds tested positive to AstV in TEM and RT-PCR. In detail, 4/5 samples tested positive to AstV in RT-PCR in Lecce 2012, 2/ 3 in Foggia 2015, 4/6 in Foggia 22,013 and 4/4 in Foggia 32,013. BLAST and FASTA analyses of the sequences confirmed the highest nt identity to the AstV strains belonging to the genus Mamastrovirus. The fecal samples were also screened for other bovine enteric pathogens. Samples of Lecce 2012 also tested positive for RVA (2/5), with sample Bov/ITA/2012/715 being a co-infection AstV + RVA. In the herd Foggia 2015, 1/3 samples also tested positive for bovine CoV, with sample Bov/ITA/2015/954-1 being a co-infection AstV + CoV. In the herd Foggia 22,013, bacteriological investigations revealed the presence of Escherichia coli, whilst in Foggia 32,013 the samples were only positive to AstV. RVA was identified in three herds, Cryptosporidium parvum and Escherichia coli in 2 different herds each (Table 1) . The sequence of~3. (Fig. 2) . Sequence comparison in the ORF1b (RdRp) was calculated using a 642 nt (214 aa) long fragment located at the very 3'end of the gene. Upon sequence comparison, Table 3 ). The similarity of the bovine with buffalo strains retrieved in this study ranged from 53.35 to 94.15% nt and from 52.00 to 93.00% aa. An 8-nt overlap, typical of bovine AstV strains, occurred between the termination codon of ORF1b and the initiation codon of ORF2 for the strains Bov/ITA/2012/715 and Bov/ITA/2015/954-1, whilst the strains Buf/ITA/2013/619 and Buf/ITA/2013/750 displayed a longer overlap of 11-nt. In the four strains, like other bovine AstVs, a cytosine replaced the initial adenosine nucleotide in the highly conserved nt stretch upstream of ORF2, ATTTGGAGNGGNGGACCNAAN 5-8 ATGNC, which is believed to be part of a promoter region for synthesis of subgenomic RNAs (Walter et al., 2001) . Also, the N 5-8 stretch was significantly longer, 11 nt for the bovine strains and 14 nt for the buffalo strains. The ORF2 of the strains Bov/ITA/2012/715, Bov/ITA/2015/954-1, Buf/ITA/2013/619 and Buf/ITA/2013/750 was 2217, 2190, 2256 and 2238 in length and encoded for a capsid precursor protein of 738, 729, 751 and 745 aa, respectively. Sequence comparison in the ORF2 (capsid) was calculated using a 2943 nt (981 aa) long fragment. Upon sequence comparison, strains Bov/ITA/2012/715 and Bov/ITA/2015/ 954-1 displayed 53.56% nt (52.31% aa) identity to each other and the highest identity to Japanese strains Ishikawa24-6 (83.54% nt, 81.79% aa) and Hokkaido12-7 (93.25% nt, 92.00% aa), respectively (Table 4) . Strains Buf/ITA/2013/619 and Buf/ITA/2013/750 showed 50.12% nt (49.27% aa) identity to each other and the highest identity to Swiss strain VC34.375 (84.20% nt, 82.55% aa) and to Chinese strain G1 (71.55% nt, 69.70% aa), respectively (Table 4 ). Identity among the Italian ruminant AstVs ranged from 51.40 to 78.81% nt and from 49.55 to 77.16% aa. The highly conserved motive SRGHAE at the C-terminus of capsid protein was not present (Jonassen et al., 1998) . The 3' UTR of the strains Bov/ITA/2012/715, Bov/ITA/2015/954-1, Buf/ITA/2013/619 and Buf/ITA/2013/750 was 72, 78, 90 and 74 nt long, respectively. Also, the conserved stem-loop II motive (s2m) conserved in several AstVs, CoVs and picornaviruses (Tengs et al., 2013) , was not present in the bovine and buffalo AstVs detected in this study. Phylogenetic tree based on the partial RdRp and complete capsid sequences showed that the four AstV strains formed distinctive cluster together with bovine, deer, buffalo, yak, ovine and porcine AstV sequences and were distantly related to human, mink, ovine and bovine neurotropic AstVs (Figs. 3 and 4) . Phylogenetic RdRp-based tree showed that strain Buf/ITA/2013/750 intermingled different clusters in which Buf/ITA/2013/619, and Bov/ITA/2013/715 segregated with bovine AstVs retrieved in Japan, USA and Switzerland between 2009 and 2017 and Bov/ITA/2015/954 segregated with Japanese bovine AstVs identified between 2009 and 2015 (Fig. 3) . Phylogenetic capsidbased tree also revealed the presence of two major clades among bovine, bubaline, ovine, deer, yak, AstV strains. In the first major clade Buf/ITA/2013/619 formed a separate cluster together with a Swiss strain VC34.375 (MK987101) (p-dist = 0.58) and Bov/ITA/2013/715 belonged to a well-defined cluster together with Japanese (Ka-goshima2-3-1, LC047797 and Ishikawa24-6, LC047787) and Chinese (B34, HQ916315) AstV strains (p dist = 0.61-0.72). Buf/ITA/2013/ 750 strain was basal to the second major clade (p-dist = 1.10-1.50) in which Bov/ITA/2015/954 formed a separate cluster with Japanese bovine AstVs (p-dist = 0.24-0.47) (Fig. 4) . In this study, we report the identification and genomic characterization of AstV strains identified from distinct outbreaks of enteritis in bovine and buffalo calves. AstVs were initially considered to be avirulent in experimental infections in calves (Woode and Bridger, 1978) . In subsequent studies, AstVs were found to elicit histological lesions in the first part of the intestine in experimental infections (Woode et al., 1985) . However, sequence data and diagnostic systems were not available until recently, thus hampering a diagnosis of AstV infection in most laboratories. Likewise, in this study the identification of AstVs was achieved by observation in TEM, even if a specific hyperimmune serum was not used to increase the sensitivity of the assay, as immunological reagents are not available for these viruses. The development of consensus primer sets for AstVs (Chu et al., 2008) finally provided researchers with a new tool for identification of diverse strains of AstVs from different animal species. Sequence and phylogenetic analysis revealed a marked genetic diversity among the four Italian ruminant AstVs that were distantly related to other AstV species officially recognized by ICTV. In the phylogenetic tree based on the partial RdRp, the four strains detected in this study formed an independent cluster together with other bovine and buffalo AstVs although strain Buf/ITA/2013/750 appeared more distantly related. Upon phylogenetic analysis based on the complete capsid region, strain Buf/ITA/2013/619 and Bov/ITA/2012/715 formed a well-defined clade with other bovine and caprine AstVs detected in Asian and European countries. In the second clade Buf/ITA/ 2013/750 was distantly related to other bovine, bubaline, yak, deer and porcine AstVs whilst Bov/ITA/2015/954-1 clustered together with bovine AstVs retrieved in Japan. In addition, all the strains sequenced in this study were highly divergent from the bovine AstV NeuroS1 and CH13 strains previously associated with neurologic disease in cattle (Li et al., 2013) . Classification of AstVs is cumbersome, due to the massive number of genetically heterogeneous strains identified from different animal species. Accumulation of AstV sequences from several animal hosts, generated in metagenomics studies, is unveiling a vast genetic diversity. The identification of animal-like AstVs in humans (De Benedictis et al., 2011; Janowski et al., 2019) and of neurotropic strains in humans and several animal species (Reuter et al., 2018) has markedly propelled the research on AstV. A 2011 revision of the ICTV classification (2009) recognized that classification based on genetic criteria is more appropriate. Based on phylogenetic analysis of MAstVs inferred on the aa sequence of the full length ORF2, 19 groups or species were proposed and the mean aa genetic distances (p-dist) range was calculated as 0.378-0.750, and as 0.006-0.312 between and within groups, respectively 1 . It is likely that with the accumulation of AstVs genome sequences, the criteria for species demarcation will be modified. Based on the aforementioned criteria, the four strains reported in this study, for instance, could represent each a candidate new species. The role of AstVs in the etiology of calves enteritis has been addressed in a limited number of epidemiological studies (Alfred et al., 2015; Nagai et al., 2015; Sharp et al., 2015) . A high prevalence of genetically diverse AstVs was reported in fecal samples from both healthy Fig. 3 . Phylogenetic tree based on partial (214aa) RdRp of astroviruses retrieved from GenBank database and bovine and bubaline astroviruses detected in this study. Posterior output of the tree was derived from Bayesian inference using 4 chains run for > 1 million generations, a general time-reversible model (6-character states), a proportion of invariable sites, a gamma distribution of rate variation across sites and a subsampling frequency of 1000. Posterior probability values > 95% are indicated on the tree nodes. Black arrows indicate the AstV strains detected in this study. Avian astrovirus strain GA2011 (GenBank accession nr JF414802) was used as an outgroup. Genus, host, country, year of detection, strain name, and GenBank accession numbers are indicated for each strain. The scale bar indicates the number of nt substitutions per site. MAstV Mammalian Astrovirus, AvAstV avian astrovirus. (caption on next page) V. Martella, et al. Research in Veterinary Science 131 (2020) 59-68 and diarrhoeic calves but no significant association between diarrhea and AstV was established (Sharp et al., 2015) . A viral metagenomics study identified 15 AstV-related RNA sequences from fecal samples from either healthy or diarrheal calves. Eleven out of these 15 AstVrelated RNA sequences were from calves with diarrhea (Nagai et al., 2015) . Screening of bovine samples from Korean cattle identified AstVs in samples from diarrheal calves (9 out 91, 9.9%) but not in 0/24 nondiarrhoeal samples (Oem and An, 2014) . Screening of bovine and buffalo herds in China has identified AstV in 92/211 samples (43.6%) (Alfred et al., 2015) . The AstV strains Bov/ITA/2012/715 and Bov/ITA/2015/954-1 were present in co-infection with bovine RVA and with CoV, respectively. Previous studies reported that bovine AstVs are not directly associated with severe diarrhea in calves under natural condition (Woode and Bridger, 1978; Bridger et al., 1984) . Eventually, infections by bovine AstV could trigger severe diarrhea in co-infections with other enteric pathogens (Woode et al., 1984; Oem and An, 2014; Nagai et al., 2015) . Overall, the discovery of a repertoire of genetically distantly related viruses from ruminants, in some case with different phenotypes (Reuter et al., 2018) , would require interpreting the literature/experiments with caution. Interestingly, in one of the water buffalo AstV-confirmed outbreak from our study, along with acute severe enteric disease, in some animals the enteric signs tended to configure as a chronic disease, with the calves showing marked delay in growth and hypotrichosis (matted and thinned fur). AstV was the only enteric pathogen detected in the buffalo herd and the problems were resolved by enacting stricter measure of prophylaxis, i.e. repeated disinfections of the calving areas and delaying the weaning phase. The bovine and buffalo AstV strains in the present study revealed marked genetic differences in the RdRp and capsid regions with respect to other ruminant AstVs, although the data generated in this study cover a relatively restricted geographical area and time span. It may be hypothesized that a vast repertoire of ruminant AstV strains exist, coupled eventually with inter-species circulation across different ruminant species. Recent discoveries and genetic and evolutionary studies of novel AstVs have suggested their potential capability to cross species barriers and adapt to new host species (Nagai et al., 2015) . Circulation of AstV among ruminants should not be uncommon, even for neurovirulent AstV strains . Our results support a scenario in which water buffalo may be one of the hosts of the AstV of bovines and/or of other related ruminants (Alfred et al., 2015; Nagai et al., 2015) . Finally, the impressive genetic diversity of ruminant AstVs pose challenges for the development of reliable and effective diagnostic algorithms for these viruses. Gathering sequence data is pivotal to refine the diagnostic tool. Also, structured surveillance studies could help decipher more precisely the overall impact of AstVs on livestock animals and conceive measure of prophylaxis. The authors declare no conflict of interest. Fig. 4 . Phylogenetic tree based on complete (981aa) capsid of astroviruses retrieved from GenBank database and bovine and bubaline astroviruses detected in this study. Posterior output of the tree was derived from Bayesian inference using 4 chains run for > 1 million generations, a general time-reversible model (6-character states), a proportion of invariable sites, a gamma distribution of rate variation across sites and a subsampling frequency of 1000. Posterior probability values > 95% are indicated on the tree nodes. Black arrows indicate the AstV strains detected in this study. Avian astrovirus strain GA2011 (GenBank accession nr JF414802) was used as an outgroup. Genus, host, country, year of detection, strain name, and GenBank accession numbers are indicated for each strain. The scale bar indicates the number of nt substitutions per site. MAstV Mammalian Astrovirus, AvAstV avian astrovirus. Molecular epidemiology and phylogenetic analysis of diverse bovine astroviruses associated with diarrhea in cattle and water buffalo calves in China Viruses and gastroenteritis in infants Detection of a novel astrovirus in brain tissue of mink suffering from shaking mink syndrome by use of viral metagenomics Indication of cross-species transmission of astrovirus associated with encephalitis in sheep and cattle Novel encephalomyelitis-associated astrovirus in a muskox (Ovibos moschatus): a surprise from the archives Neurotropic astrovirus in cattle with nonsuppurative encephalitis in Europe Characterization of a calici-like virus (Newbury agent) found in association with astrovirus in bovine diarrhea The electron microscopical and physical characteristics of small round human fecal viruses: An interim scheme for classification Novel astroviruses in insectivorous bats Astrovirus infections in humans and animals -molecular biology, genetic diversity, and interspecies transmissions Detection of bovine coronavirus using a TaqMan-based real-time RT-PCR assay Concentrating rotaviruses from water samples using monolithic chromatographic supports MRBAYES: Bayesian inference of phylogenetic trees Differential in vitro infection of neural cells by astroviruses A common RNA motif in the 3′ end of the genomes of astroviruses, avian infectious bronchitis virus and an equine rhinovirus MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform MEGA X: molecular evolutionary genetics analysis across computing platforms Divergent astrovirus associated with neurologic disease in cattle Development of a TaqMan assay for sensitive detection of all pestiviruses infecting cattle, including the emerging HoBi-like strains Astroviruses Astroviruses Pathogenesis of astrovirus infection Full genome analysis of bovine astrovirus from fecal samples of cattle in Japan: identification of possible interspecies transmission of bovine astrovirus Phylogenetic analysis of bovine astrovirus in Korean cattle A novel astrovirus associated with encephalitis and ganglionitis in domestic sheep Selection of models of DNA evolution with jModelTest Astrovirus encephalitis in boy with X-linked agammaglobulinemia Nonsuppurative (aseptic) Meningoencephalomyelitis associated with neurovirulent astrovirus infections in humans and animals MrBayes 3: Bayesian phylogenetic inference under mixed models Detection of a novel bovine astrovirus in a cow with encephalitis 3′ end cDNA amplification using classic RACE Frequency and pathological phenotype of bovine astrovirus CH13/neuroS1 infection in neurologically-diseased cattle: towards assessment of causality Identification of a second encephalitis-associated astrovirus in cattle High prevalence and diversity of bovine astroviruses in the faeces of healthy and diarrhoeic calves in south West Scotland A mobile genetic element with unknown function found in distantly related viruses Rediscovery and genomic characterization of bovine astroviruses Novel human astroviruses: novel human diseases? Molecular characterization of a novel recombinant strain of human astrovirus associated with gastroenteritis in children Isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves Astrovirus and Breda virus infections of dome cell epithelium of bovine ileum Serotypes of bovine astrovirus Molecular characterization of cryptosporidium oocysts in samples of raw surface water and wastewater Prevalence and genetic characterization of caliciviruses among children hospitalized for acute gastroenteritis in the United States The study was founded with grants from Ricerca Corrente 2013 of Italian Ministry of Health "IZS PB 07/13 RC".