key: cord-0742184-k5u640q3 authors: Luo, Zhiyao; Roi, Stéphanie; Dastor, Margaux; Gallice, Estelle; Laurin, Marc-André; L’Homme, Yvan title: Multiple novel and prevalent astroviruses in pigs date: 2011-05-05 journal: Veterinary Microbiology DOI: 10.1016/j.vetmic.2010.11.026 sha: e2ac513c77cc86808fdcf4ddbfe9be0fd1344690 doc_id: 742184 cord_uid: k5u640q3 Abstract Knowledge of porcine astrovirus diversity and epidemiology remains limited. We used a broad range PCR approach to investigate the presence and diversity of astroviruses in healthy pigs of different ages on 20 farms and in 3 slaughterhouses situated in the province of Quebec, Canada between 2005 and 2007. Our study unexpectedly revealed remarkable levels of genetic diversity and high prevalence of astroviruses in pigs of this province. Astroviruses were detected on every farm investigated and in all age groups of pigs, from suckling piglets to adults. In addition, we found that nearly 80% of healthy finisher pigs harbour astroviruses in their intestine at slaughter. Phylogenetic evidence based on partial polymerase and complete capsid sequences, suggests that porcine astroviruses do not form a monophyletic group but are rather found on separate branches across the mamastrovirus tree. In addition to type species strains, we found highly divergent strains that form two additional lineages, one of which falls outside existing taxonomic groups. The presence of diverse astroviruses in a majority of healthy pigs likely represents a continuous source of infection to piglets and possibly to other animal species including humans. Porcine astrovirus strains appeared phylogenetically related not only to prototypical human astroviruses, as was already known, but also to novel human strains recently discovered suggesting multiple cross species transmission events between these hosts and other animal species. Overall, the findings reported in this study suggest an active role of pigs in the evolution and ecology of the Astroviridae. The family Astroviridae consists of small (28-30 nm), non-enveloped, single-stranded positive sense RNA viruses of 6.4-7.3 kb in size. These viruses generally exhibit a distinctive five or six pointed star shape appearance under EM which led Madeley and Cosgrove (1975) to coin the term astrovirus to describe them. The AstV genome includes three open reading frames (ORFs) designated ORF1a, ORF1b and ORF2. ORF1a and ORF1b situated at the 5 0 end of the genome encode non-structural polyproteins including a protease and a RNA dependent RNA polymerase (RdRp) . ORF2 situated at the 3 0 end of the genome encodes the structural capsid protein and is transcribed as a subgenomic mRNA (Mendez and Arias, 2007; Monroe, 2005) . Astroviruses are enteric viruses that have been isolated from a number of host species including humans, mink, sheep, pigs, chicken, ducks, turkeys and more recently from marine mammals, dogs, cheetahs and bats (Monroe, 2005; Jonassen et al., 2001; Zhu et al., 2009; Rivera et al., 2010; Atkins et al., 2009; Toffan et al., 2009) . The family Astroviridae is currently divided in two genera; strains of the genus Mamastrovirus are found in infected mammals and strains of the genus Avastrovirus are found in avian hosts (Monroe, 2005) . According to the latest ICTV report, Knowledge of porcine astrovirus diversity and epidemiology remains limited. We used a broad range PCR approach to investigate the presence and diversity of astroviruses in healthy pigs of different ages on 20 farms and in 3 slaughterhouses situated in the province of Quebec, Canada between 2005 and 2007. Our study unexpectedly revealed remarkable levels of genetic diversity and high prevalence of astroviruses in pigs of this province. Astroviruses were detected on every farm investigated and in all age groups of pigs, from suckling piglets to adults. In addition, we found that nearly 80% of healthy finisher pigs harbour astroviruses in their intestine at slaughter. Phylogenetic evidence based on partial polymerase and complete capsid sequences, suggests that porcine astroviruses do not form a monophyletic group but are rather found on separate branches across the mamastrovirus tree. In addition to type species strains, we found highly divergent strains that form two additional lineages, one of which falls outside existing taxonomic groups. The presence of diverse astroviruses in a majority of healthy pigs likely represents a continuous source of infection to piglets and possibly to other animal species including humans. Porcine astrovirus strains appeared phylogenetically related not only to prototypical human astroviruses, as was already known, but also to novel human strains recently discovered suggesting multiple cross species transmission events between these hosts and other animal species. Overall, the findings reported in this study suggest an active role of pigs in the evolution and ecology of the Astroviridae. Crown Copyright ß 2010 Published by Elsevier B.V. All rights reserved. the Avastrovirus genus contains 3 species while the Mamastrovirus genus contains 6 species, all of which are defined based on the species of origin (Monroe, 2005) . Astroviruses are generally associated with either mild or severe signs of enteric disease such as diarrhoea and vomiting in a number of mammalian species (Mendez and Arias, 2007) . Astroviruses have also been found to cause no detectable disease or to cause disease only occasionally (Bridger et al., 1984; Marshall et al., 1987; Chu et al., 2008) . Human astroviruses (HuAstVs) have been the most extensively studied group of astroviruses and have been repeatedly shown to be a common cause of diarrhoea in young children, the elderly and the immunocompromised (Dennehy et al., 2001; Gallimore et al., 2006; Gray et al., 1987; Akihara et al., 2005) . AstV also appear second to rotaviruses as gastroenteritis agents responsible for children hospitalization (Glass et al., 1996) . There are presently 8 recognized serotypes of closely related HuAstVs, all of which belong to a single species (Monroe, 2005) . Phylogenetic groupings (genotypes) of HuAstV strains were found to be fully congruent with serotyping analyses since 8 well defined clusters were formed regardless of the genomic region analysed (Noel et al., 1995; Lukashov and Goudsmit, 2002) . In recent years, a number of genetically distinct strains of HuAstV have been identified, characterized and proposed to represent novel AstV species based on a genetic distance criteria (Finkbeiner et al., 2008; Kapoor et al., 2009) although this proposal is still awaiting ICTV's approval. Little is known about the presence, clinical significance and diversity of porcine astroviruses. Porcine AstVs were first detected by EM in faeces of a diarrheic piglet (Bridger, 1980) . It was then successfully propagated in porcine embryonic kidney cells and shown to cause mild diarrhoea in piglets under experimental conditions (Shimizu et al., 1990) . Molecular characterization and phylogenetic analysis of the capsid gene from this isolate (PAstV-1) followed and helped determine that past spill over events between cats, pigs and humans were likely to have happened (Jonassen et al., 2001; Wang et al., 2001) . A closely related strain was also more recently isolated from a pig diarrhoeal sample and successfully propagated in vitro (Indik et al., 2006) . Apart from these few reports, there is to knowledge no available data on the diversity of porcine astroviruses. We used a PCR approach to investigate the presence and diversity of porcine astroviruses using both specific and broad range (pan-astrovirus) primers. Our study reveals the presence in swine of prototypical strains as well as two lineages of previously unknown astroviruses. In addition, we found that astroviruses were present on all swine farms investigated independent of age group and in nearly 80% of slaughtered healthy pigs. A total of 200 composite faecal samples were collected from 20 farms in the province of Quebec, Canada between 2005 and 2007. Each sample consisted of a mix made up of 5 sub samples collected in different parts of each pen. 60 samples were from nursing pigs (less than 4 weeks of age), 60 samples were from post-weaning pigs (between 4 and 12 weeks of age) and 80 samples were from finisher pigs (over 12 weeks of age). A total of 96 faecal samples originating from individual caecal content of slaughtered adult pigs in 3 different abattoirs in the province of Quebec were also collected during the same period. Viral RNA was extracted from the supernatants of 20% (w/v) faecal suspensions in Eagle's minimum essential medium (MEM, Invitrogen, Mississauga, ON, Canada) using the viral RNA mini kit (Qiagen, Mississauga, ON, Canada). 140 ml of the suspension was treated following the manufacturer's protocol. The RNA was stored at À70 8C until needed. All primers used in this study are listed in Table 1 and were synthesized at Integrated DNA Technologies (IDT, Coralville, IA, USA). Preliminary screenings were performed separately with two sets of primers targeting different regions of the AstV genome using the one-step RT-PCR kit from Qiagen (Mississauga, ON, Canada). AST248F/AST665R primer pair was originally designed based on a conserved region at the 5 0 end of open reading frame 2 (ORF2) encoding the capsid protein of a porcine astrovirus strain (Indik et al., 2006) . A volume of 1 ml of the RNA extract was mixed with 5 ml of 5Â buffer, 400 nM of each deoxinucleotide triphosphate (dNTP), 400 nM of each primer and 1 ml of the enzyme mix in a final volume of 25 ml. Following the initial RT reaction at 42 8C for 30 min, the reaction was denatured at 95 8C for 15 min and amplified for 40 cycles of the following conditions: 94 8C for 30 s, 55 8C for 30 s and 72 8C for 30 s. A final extension was done at 72 8C for 5 min. Semi-nested PCR was performed using 1 U of Platinum Taq DNA polymerase (Invitrogen, Mississauga, ON, Canada) in a final reaction volume of 50 ml containing 1 ml of a 1/1000 diluted cDNA, 3 mM MgCl 2 , 200 nM each dNTP, 200 nM of forward (ASTneF) and reverse primer (ASTneR). The reaction mix was initially denatured at 94 8C for 2 min and amplified for 25 cycles with the following conditions: 94 8C for 15 s, 55 8C for 30 s, 72 8C for 30 s and a final extension at 72 8C for 7 min. The primers were originally designed based on a conserved region in the RNA-dependent-RNA polymerase (RdRp) gene of AstVs (Chu et al., 2008) . PCR primers panAV-F11 (forward), panAV-F12 (forward) and panAV-R1 (reverse) were used in the initial RT-PCR; while primers panAV-F21 (forward), panAV-F22 (forward) and panAV-R1 (reverse) were used in the semi nested PCR. 1 ml of RNA extract was mixed with 5 ml of 5Â buffer, 400 nM of each deoxinucleotide triphosphates (dNTP), 400 nM of each primer and 1 ml of enzyme mix in a final volume of 25 ml. Following the initial RT reaction at 50 8C for 60 min, the reaction was denatured at 95 8C for 15 min and amplified for 40 cycles of the following conditions: 94 8C for 30 s, 50 8C for 30 s and 72 8C for 30 s with a final extension at 72 8C for 5 min. Semi-nested PCR was performed using 1 U of Platinum Taq PCR products were either processed on standard 1% agarose gels and stained with SYBR safe (Invitrogen, Mississauga, ON, Canada) or analysed using a Qiaxcel instrument (Qiagen, Mississauga, ON, Canada). Expected amplicon sizes were approximately 180 bp using nested Fig. 1 . Phylogenetic relationship of Canadian porcine astroviruses (boxed) and prototypical astrovirus species. (a) Tree based on partial RdRp coding region ($300 nt) amplified using pan-astrovirus primers. (b) Tree based on complete ORF2 coding region ($800 aa). Phylogenetic trees were generated using the neighbour-joining method implemented in the program MEGA4. Bootstrap values are expressed in percentage based on 1000 replications and indicated at the node. Bootstrap values of 70% were hidden. Scale bar represents distance expressed as nucleotide or amino acid substitutions per site. Accession numbers for the AstV strains characterized in this study are from HM756258 to HM756273. PCR 1 or 400 bp using nested PCR 2. PCR products of the expected size were either directly sequenced in both directions using the big dye v3.1 chemistry on a 3730xl instrument from Applied Biosystems (Foster, CA) or cloned using the TOPO 2.1 (T/A) cloning (Invitrogen, Mississauga, ON). A minimum of three clones from each strain was sequenced in the latter case. 2.3. Amplification of the 3 0 end of selected AstV samples using 3 0 RACE-PCR Based on consensus nucleotide sequences of the RdRp fragments obtained from strains of the 3 different groups of porcine astroviruses, group specific primers were designed using Primer3 software (http://frodo.wi.mit.edu/cgibin/primer3/primer3_www.cgi). Specific strains were selected as representatives of each of the 3 porcine AstV groups: PoAstV12-3 for group I, PoAstV12-4 and PoAstV14-4 for group II and PoAstV16-2 for group III (see Fig. 1 ). The group-specific primers listed in Table 1 were used in combination with primers (QT), (QO) and (QI) (Scotto-Lavino et al., 2006) as previously described (L'Homme et al., 2009 ) for amplification of the 3 0 ends of the selected porcine AstV strains. cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen, Mississauga, ON, Canada) and primer QT at 50 8C for 60 min following the manufacturer's instructions. A first PCR was performed with primers QO and strain-specific F1 primers using 1 U of Platinum Taq The RACE-PCR products were deposited on 1% agarose gels, stained with SYBR safe (Invitrogen, Mississauga, ON, Canada) and purified on Qiagen mini spin columns (Qiagen, Mississauga, ON, Canada). Gel purified amplicons were cloned using the PCR TOPO 2.1 (T/A) cloning kit (Invitrogen, Mississauga, ON, Canada). A minimum of three clones were selected for each strain, amplified in liquid broth followed by plasmid DNA extraction using an alkaline lysis method (QIAprep Spin Miniprep, Qiagen). Each clone was then completely sequenced in both directions using a primer walking strategy. Sequence editing, assembly and analysis were performed using BioEdit version 7.0.9.0 (http://www.mbio.ncsu.edu/ bioedit/bioedit.html). Multiple sequence alignments were done with CLUSTAL W (version 1.6). Phylogenetic analyses were carried out using the neighbour-joining (NJ) method with the Poisson distance correction calculation for amino acids and maximum composite likelihood model for nucleotides using the Molecular Evolutionary Genetic Analysis (MEGA 4.0) software. Percentage identity of the capsid gene was calculated using amino acid p-distance implemented in MEGA 4.0. Confidence values at the nodes were obtained by performing 1000 bootstrap analyses. In our preliminary screening of farm composite faecal samples, we used two different sets of nested primers. The first set (primer set 1) targets a conserved region at the 5 0 end of ORF2 and was specifically designed for and reported to successfully amplify porcine astroviruses (Indik et al., 2006) . To maximize the likelihood of detecting a wide range of variable AstV strains, a second set (primer set 2) of degenerated primers was used (pan-astrovirus primers) which was designed based on conserved motifs from the AstV RdRp gene (Chu et al., 2008) . This pan-astrovirus primer set has been shown to successfully detect a wide array of variable astroviruses in humans (Kapoor et al., 2009 ) and bats (Chu et al., 2008; Zhu et al., 2009) . Selected amplicons were either sequenced directly in both directions or cloned and sequenced depending on the amount of DNA amplified and/or on the presence of multiple amplicons of similar sizes on agarose gels. Pairwise identity comparisons of deduced amino acid sequences amplified using primer set 1 revealed that all porcine AstV sequences were highly similar to each other and to prototypical porcine strain PAstV-1 (Genbank accession # Y15938) (>97% amino acid identity). Phylogenetic analyses using amino acid or nucleotide sequences formed a monophyletic group (not shown). Alignment of the deduced amino acid sequences amplified using the pan-astrovirus primers (primer set 2) revealed that all porcine strains amplified contained the characteristic ''YGDD'' motif located near the C-terminal region of the RdRp (not shown). This motif is found in the RdRp protein of all astroviruses characterized so far as well as in the RdRp of other RNA virus families such as the Caliciviridae (Green, 2007) . Pairwise identity comparisons of deduced amino acid sequences amplified using primer set 2 revealed more heterogeneity among strains (51-98% amino acid identity) than sequences generated using primer set 1 even though the former are situated in the more highly conserved polymerase region. Phylogenetic analyses using these partial polymerase sequences from selected strains in addition to prototypical animal AstV strains clustered Canadian porcine strains in three distinct lineages (Fig. 1a -group I, II, III). Group I strains were related to PAstV-1 strain and to typical human and feline strains. Group II strains were isolated on a separate branch from other known AstVs with no apparent closely related strains. Finally, group III strains were related to mink, ovine and most interestingly to novel human AstV strains. A total of 4 strains (PoAstV12-3, PoAstV12-4, PoAstV14-4, and PoAstV16-2) were selected as representatives of each of the three clusters of PoAstVs to permit in depth genomic investigation and strengthen their taxonomic grouping. Using 3 0 RACE-PCR, we were able to amplify and characterize approximately 3 kb of nucleotide sequence from each strain which included the 3 0 end of the RdRp gene, the complete capsid gene and the 3 0 UTR (Fig. 2a) . Astroviruses contain a conserved sequence (UUUG-GAGNGGNGGACCNAAN 5-8 AUGNC) located at the junction between ORF1b and ORF2 which has been proposed to be a regulatory element serving as a promoter for subgenomic RNA transcription (following the convention of HuAstV which places the initiation AUG codon -underlined -for ORF2 immediately upstream of the ORF1b stop codon) (Mendez and Arias, 2007) . Alignment of the corresponding region from the 4 representative PoAstV strains characterized here along with prototypical AstV strains revealed that it is highly conserved in all strains (shown for $30 nt in Fig. 2b) . Interestingly, strains PoAstV12-4 and PoAstV14-4 contain a unique insertion of 3 nucleotides upstream of the ORF2 initiation codon which is not present in any of the other AstV strains. The ORF2 start codon (bold) of strains PoAstV12-3 and PoAstV16-2 appeared in an optimal Kozak context for translation initiation (RNNAUGG, where R = A/G and N = A/T/G/C) (Kozak, 1991) , whereas those of strains PoAstV12-4 and PoAstV14-4 appeared in a less favourable context with a C residue at position -3 (Fig. 2b) . The ORF2 genes are predicted to encode capsid proteins of between 755 and 783 amino acids in length which is comparable to all mamastrovirus known to date (Table 2) . Molecular masses (Mr) are predicted to be between 82.1 kDa and 85.6 kDa. The length of the 3 0 UTR of the four PoAstV strains varied from 76 to 86 nucleotides which is comparable to most mamastrovirus known to date ( Table 2 ). All mamastrovirus strains except human strain MLB-1 contain a highly conserved stem-loop II-like motif (s2m) in the 3 0 UTR. This motif has also been reported in some picornaviruses and coronaviruses (Jonassen et al., 1998) . The exact role of this motif is unclear, but due to its presence in many viral families, it has been suggested to have an important function in viral RNA replication. As is shown in Fig. 2c , alignment of this region reveals that strains PoAstV12-3 and PoAstV16-2 contain a highly conserved s2m motif whereas the corresponding sequence of strains PoAstV12-4 and PoAstV14-4 appear highly divergent, which is reminiscent of human strain Ast-MLB-1 (Finkbeiner et al., 2008) . Phylogenetic and pair-wise amino-acid identity analyses of the complete capsid coding region confirmed the close relationship of strain PoAstV12-3 with prototypical PAstV-1 strain (80% aa identity) ( Fig. 1b and Table 3 ). Strain PoAstV16-2 appeared divergent from prototypical porcine strains (28% aa identity) and closest to mink AstV (58% aa identity) and human HMO strain (54% aa identity) (Table 3 and Fig. 1b) . Most interestingly, phylogenetic and distance analyses revealed that strains PoAstV12-4 and PoAstV14-4 were highly divergent from known AstV strains and formed a novel group of mammastrovirus distantly related to the human/feline/porcine AstV group (Fig. 1b and Table 3 ). Altogether, these analyses confirmed that swine harbour phylogenetically diverse AstV strains most likely derived from distinct ancestors. Phylogenetic analyses performed with either the larger polymerase fragment ($250 aa) (not shown) or the complete capsid coding sequence from the 4 novel porcine strains did not reveal any differences in the branching patterns of the threes indicating that these strains are unlikely to represent recombinants. Using both primer sets 1 and 2, we were able to detect and confirm the presence of astroviruses on every farm and in all age groups from suckling piglets to finisher pigs. Since the pan-astrovirus primers detected a large number of AstV samples that were undetected by primer set 1, we used the former to screen a sample set from ceacal contents of 96 individual slaughtered finisher pigs. 76 Table 2 Genome features comparison of novel porcine AstV from this study and selected reference strains. ORF1b-ORF2 overlap (nt) ORF2 length (aa) 3 0 UTR length (nt) Accession number Table 3 Pairwise amino acid (aa) sequence identity between selected AstV strains. 3 53 53 47 87 83 87 85 NA 81 64 52 47 49 52 33 PoAstV 12-4 30 94 48 52 51 52 49 NA 58 54 45 49 53 57 36 PoAstV 14-4 31 60 45 52 51 52 51 NA 59 52 47 46 44 54 35 PoAstV 16-2 29 23 24 46 48 46 45 NA 44 46 65 62 59 68 32 Human AstV-1 50 30 32 27 95 100 97 NA 80 62 51 46 51 52 30 Human AstV-3 52 32 33 28 81 93 NA 77 62 49 45 48 52 31 Human AstV-4 47 30 32 27 73 74 97 NA 80 62 51 46 51 52 30 Human AstV-6 48 30 32 28 78 80 74 NA 78 62 49 45 51 51 30 Porcine AstV 80 30 31 28 50 51 46 48 NA NA NA NA NA NA NA Dog AstV 42 29 28 30 40 41 40 39 42 65 47 45 52 49 38 AstV-MLB 31 27 28 25 32 30 30 29 32 29 46 52 47 47 33 Bat AstV 30 25 25 34 30 30 28 30 29 28 26 63 64 67 31 Ovine AstV 31 25 26 50 29 29 29 29 30 31 25 34 62 64 33 Mink AstV 28 23 26 58 27 27 27 28 28 32 26 33 52 68 30 HMO AstV-2 28 25 25 54 28 28 27 27 27 29 26 33 51 51 38 Turkey AstV-1 20 18 18 21 19 20 20 19 20 22 21 18 28 28 28 Values above and below the diagonal indicate partial ORF1b ($250 aa) identity and complete ORF2 identity respectively. Values underlined indicate interstrain identities between the 4 porcine strains characterized in this study and values in boldface indicate identities between the 4 newly characterized porcine strains and the prototypical porcine strain (Y15938). NA: not available. samples (79%) were confirmed positive by sequencing of the nested PCR product. Phylogenetic analyses clustered 1% with group I, 99% with group II and none with group III. Strains from group III were only detected in farm samples from suckling piglets. We were also able to confirm a double infection with two different AstV strains in a single pig at slaughter. Attempts in culturing strains from the three PoAstV groups in PK-15 cells with various concentrations of trypsin (2-9 mg/ml) were unsuccessful. Also, direct examination of faecal samples by EM did not permit visualization of typical star shape virions of the expected size although viral particles of the expected sizes were frequently observed (25-30 nm) (not shown). This report presents the first comprehensive investigation on the presence and diversity of AstVs in pigs. A remarkable and surprising diversity of AstVs was disclosed in these hosts. Based on the data presented, which includes genomic and phylogenetic analyses, we conclude in the existence of three distinct lineages of porcine astroviruses, two of which are unprecedented. Pairwise amino acid identities between strains within each of these lineages range from only 23% to 31%, which is comparable to distances between established AstV species. Porcine strains belonging to two of the lineages (group I and III) were more closely related to other animal's AstV strains than they were to each other suggesting different ancestral origin of porcine AstVs and past interspecies transmission involving sheep, mink, felines, humans and possibly additional yet unknown hosts. Likewise, a number of novel human AstV strains were recently shown to be genetically closer to animal AstVs strains and more distantly related to type HuAstV species suggesting different origins (Finkbeiner et al., 2008; Kapoor et al., 2009 ). In addition, Chu et al. (2008) and Zhu et al. (2009) have also shown that AstVs isolated from the same species of bats cluster in different groups following phylogenetic reconstruction. Taken together, data from these three mammalian hosts suggest that AstVs found within a single animal species can have distinct origins most likely reflecting past interspecies transmission which underscores the zoonotic potential of these viruses. According to the latest ICTV guidelines, AstV species are defined based on the host of origin (Monroe, 2005) . Hence, reminiscent of a number of AstV strains recently recovered from humans and bats (Kapoor et al., 2009; Chu et al., 2008) , the PoAstV strains characterized in this study should be classified as a single viral species. However, it is now clear that multiple AstV lineages can infect the same animal species and there is also growing evidence that AstVs can cross the species barrier (Chu et al., 2008; Zhu et al., 2009; Kapoor et al., 2009) . For these reasons, it has been suggested that the classification of AstV should not be solely based on the species of origin but should also rely on a distance criteria of either the RdRp or the capsid genes (Kapoor et al., 2009) . The data presented in the present study supports this contention. Porcine strains from one of the lineages (group I) were closely related to typical swine strains which have previously been characterized and associated with diarrhoea in pigs (Jonassen et al., 2001; Indik et al., 2006) . Since all samples from this study, including those from group I, were from apparently healthy pigs, our results shed a new light on the pathogen/host relationship between pigs and astroviruses. It is well known that AstVs can cause enteric disease in numerous animal species (Mendez and Arias, 2007; Toffan et al., 2009; Atkins et al., 2009 ). However, it has also been reported that asymptomatic carriers of AstV exist (Rivera et al., 2010; Kapoor et al., 2009; Chu et al., 2008) . AstV infections in swine may express their pathogenicity in only a small percentage of cases, in conjunction with other pathogens or more severely in young animals like has often been observed in other animal species such as humans (Madeley and Cosgrove, 1975; Bridger, 1980; Woode and Bridger, 1978; Snodgrass and Gray, 1977) . It should be noted that only two strains of group I AstVs were detected in our study and that mild diarrhoea could have been overlooked in those cases. Alternatively, as mentioned before, there is a possibility that AstVs might express their pathogenicity in only a small percentage of cases. Porcine strains belonging to group II formed a lineage that appeared phylogenetically isolated from previously characterized AstV strains. Strains of this group (PoAstV12-4 and PoAstV14-4) also contained divergent nucleotide sequences in both the ORF1b/ORF2 consensus region and in the 3 0 UTR region (s2m motif) that are usually highly conserved among the Astroviridae which emphasizes the distinctness of strains belonging to this lineage. This situation is reminiscent of some human, bat and turkey strains (Jonassen et al., 1998; Chu et al., 2008; Finkbeiner et al., 2009) . These findings bring into question the biological role of the s2m motif which is thought to be important in the viral life cycle (Jonassen et al., 1998 (Jonassen et al., , 2001 . Perhaps these divergent strains are able to compensate by forming stable secondary structures with a similar role to the s2m in different parts of their 3 0 UTR. Unlike strains from group I and III, strains from group II were not closely related to other animal's strains known to date and might represent an AstV group restricted to swine. A third lineage of porcine strains (group III) appeared phylogenetically related to mink, ovine and novel human AstV strains, again suggesting past interspecies transmission events between these animals. Interestingly, strains from group III were only detected in suckling piglets which might suggest an age restricted pattern of infection for these strains. We found high infection rates of AstVs in finisher pigs. According to our nested PCR results, 80% of the animals harbour astroviruses in their intestine. Our data corroborates previous reports where seroprevalence to AstV in pigs as high as 83% were reported in some herds (Shimizu et al., 1990) . Group II PoAstVs were by far the most commonly detected AstV strains in the sampled population using the pan-AstV approach suggesting it was the dominant group of AstV in the studied population. Such high virus detection rates could imply that pigs are persistently infected with various strains of AstVs similar to what has been reported in bats (Chu et al., 2008) . Further studies to determine the pathogenic potential of porcine AstVs belonging to the different lineages in pigs of different ages are warranted. This work unveiled extensive diversity and high prevalence of AstVs in porcine hosts. Genetic and phylogenetic characterization of the complete 3 0 end from selected porcine AstV strains revealed the existence of 3 distinct lineages, most likely of different origins, with genetic distances between these being consistent with distances found between established species in the family Astroviridae. The fact that two lineages of porcine AstVs are phylogenetically related to human strains underscores the zoonotic potential of theses viruses. In addition, the work also showed high prevalence of AstVs in healthy pigs of different ages bringing into light new data about the relationship between porcine AstVs and their host. The combination of a large and diverse gene pool in pigs makes these animals an important and pivotal reservoir for the evolution and ecology of the Astroviridae. Existence of multiple outbreaks of viral gastroenteritis among infants in a day care center in Japan Characterization of an outbreak of astroviral diarrhea in a group of cheetahs (Acinonyx jubatus) Detection by electron microscopy of caliciviruses, astroviruses and rotavirus-like particles in the faeces of piglets with diarrhoea Characterization of a calici-like virus (Newbury agent) found in association with astrovirus in bovine diarrhea Novel astroviruses in insectivorous bats A prospective case-control study of the role of astrovirus in acute diarrhea among hospitalized young children Complete genome sequence of a highly divergent astrovirus isolated from a child with acute diarrhea Detection of newly described astrovirus MLB1 in stool samples from children Environmental monitoring for gastroenteric viruses in a pediatric primary immunodeficiency unit The changing epidemiology of astrovirus-associated gastroenteritis: a review An outbreak of gastroenteritis in a home for the elderly associated with astrovirus type 1 and human calicivirus Caliciviridae: The Noroviruses Isolation and partial characterization of a novel porcine astrovirus A common RNA motif in the 3 0 end of the genomes of astroviruses, avian infectious bronchitis virus and an equine rhinovirus Comparison of capsid sequences from human and animal astroviruses Multiple novel astrovirus species in human stool Structural features in eukaryotic mRNAs that modulate the initiation of translation Genetic diversity of porcine Norovirus and Sapovirus: Canada Evolutionary relationships among Letter: 28 nm particles in faeces in infantile gastroenteritis Virus and virus-like particles in the faeces of cats with and without diarrhoea Astroviruses Astroviridae Typing of human astroviruses from clinical isolates by enzyme immunoassay and nucleotide sequencing Characterization of phylogenetically diverse astroviruses of marine mammals 3 0 end cDNA amplification using classic RACE Cytopathic astrovirus isolated from porcine acute gastroenteritis in an established cell line derived from porcine embryonic kidney Detection and transmission of 30 nm virus particles (astroviruses) in faeces of lambs with diarrhoea Genetic characterization of a new astrovirus detected in dogs suffering from diarrhoea Genetic analysis of the capsid region of astroviruses Isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves Detection of diverse astroviruses from bats in China This work was supported by the Science Division of the Canadian Food Inspection Agency (CFIA). The authors extend their gratitude to Carole Simard for her constant and unconditional support.