1 Unité de Génétique Virale et Biosécurité, AFSSA, BP 5035, 22440 Ploufragan, France
2 Unité d'Epidémiologie Porcine, AFSSA, BP 5035, 22440 Ploufragan, France
3 Unité des Virus Emergents, EA 3292, Établissement Français du Sang, Bd Baille, 13005 Marseille, France
Correspondence
L. Bigarré
l.bigarre{at}ploufragan.afssa.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ811915AJ811938.
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MAIN TEXT |
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Other anelloviruses have been partially or fully characterized in several animal species, including domestic animals, such as dogs, cats, pigs, bovines, chickens and ovines, as well as wild animals, such as tupaias (tree shrews) and non-human primates (Cong et al., 2000; Leary et al., 1999
; Okamoto et al., 2001b
, 2002
; Verschoor et al., 1999
). Interestingly, the genome of anelloviruses tends to be smaller when the order of the infected animal is lower, varying from 3·8 kb for human viruses to 2·2 kb for tupaia viruses and reaching 2·9 kb for the only available full-length viral genome from a pig (Sus domesticus) in Japan (tentatively named TTV-Sd31) (Okamoto et al., 2002
). Although less exhaustive than for humans, studies suggest high natural prevalences of anelloviruses in some animals, as well as some intra- and inter-individual genetic variability (Okamoto et al., 2001b
; Thom et al., 2003
; Verschoor et al., 1999
). Recently, viral prevalences ranging between 33 and 100 % have been reported in the sera of pigs from Canada, China, Korea, Spain, Thailand and the USA (McKeown et al., 2004
).
Here, we report the first molecular survey of pig anelloviruses in herds in Brittany (France). This study was conducted in order to estimate the prevalence and diversity of the virus genetic pool and its associated risks. Initially, samples were collected in pig herds all over Brittany, France, between July 2000 and January 2002, for an epidemiological study of another member of the family Circoviridae, Porcine circovirus 2 (PCV2) (de Boisséson et al., 2004). Among the available samples, a series was chosen randomly for the search for anelloviruses. From 14 herds [named ht and v (de Boisséson et al., 2004
)], one to three animals (total 32, named with a Greek letter) were analysed for virus presence in five tissue samples (tonsil, ileum, lung and inguinal and mesenteric lymph nodes). In an additional herd (herd w), a single organ (lung) was collected from a piglet that was killed in 2003. Tissues were collected and stored at 20 °C until DNA extraction and PCR analysis.
Total DNA was extracted by using a DNeasy tissue kit (Qiagen) from 20 mg frozen tissue. DNA was resuspended in 20 µl buffer. For PCR, the two primers COM-1sens (5'-CRSWKMCGAATGGYWGAGTTTWY-3') and COM-2rev (5'-GCCCGAATTGCCCCTWGACTKCG-3'), which targeted two conserved domains within the UTR, were used (Biagini et al., 2003). A 2 µl aliquot of DNA was used in a reaction volume of 50 µl containing 90 pmol each primer, 1·5 mM MgCl2 and 2 U Taq polymerase (Eurobio). An initial step of 4 min at 94 °C was performed, followed by 38 cycles of 94 °C for 1 min, 57 °C for 30 s, 72 °C for 20 s and a final elongation step of 5 min at 72 °C. A 20 µl aliquot of the reaction product was electrophoresed on a 3 % TAE/agarose gel stained with ethidium bromide. DNA bands of the expected size (about 115 bp) were gel-purified and TA-cloned in pCR4-TOPO (Invitrogen). Sequencing was performed on both strands by using an ABI Prism dye terminator cycle sequencing kit and an ABI Prism 3100 Avant sequence analyser (Perkin Elmer). Between two and ten bacterial clones were sequenced for each cloned PCR product. Series of negative controls (water) were performed repeatedly for each amplification, to ensure the absence of contamination.
Nucleotide sequences were edited with the package Vector NTI Advance (Informax) and aligned with the CLUSTAL W method implemented in the package. Identity levels were obtained by simultaneously aligning all of the representative sequences produced here and anellovirus sequences from other isolates available in GenBank. Phylogenetic analysis of aligned sequences was performed by using the neighbour-joining method with the TamuraNei model, implemented in the MEGA3 program (Kumar et al., 2004
; available at http://www.megasoftware.net/mega3/). A bootstrap resampling analysis of 500 replicates was performed.
Anelloviruses were detected in 93 % (14/15) of the herds. Only herd r, in which two animals were tested, was negative. The frequency of animals that were positive for at least one organ was 73 % (24/33). When an individual was found to be positive, not all of its organs necessarily gave a PCR product. For instance, in herds hv, only one animal was positive for all five organs, whereas about one-third (10/32) of the animals had one positive organ (three positive organs, 21·9 %; four organs, 9·3 %; two organs, 6·2 %). In total, 32·5 % (52/160) of all tested organs were positive, the lung being the most frequently positive organ (lung, 11·2 %; inguinal lymph node, 8·1 %; mesenteric lymph node, 5·6 %; tonsil, 4·4 %; ileum, 3·1 %).
For each animal that exhibited at least one positive organ, one PCR product was cloned and a few (two to ten) clones were sequenced. In total, 82 sequences were obtained that, once aligned, were grouped in a total of 24 variants that exhibited between 78 and 99 % identity to one another. The sequences varied between 59 and 78 bp, differing by point mutations combined (or not) with insertions/deletions (Fig. 1). However, it cannot be excluded that some variants, unique by one single base, may have arisen from PCR artefacts, despite the small size of the amplicon. All of our viral clones showed the motif GMCTGGGCGGGTGCCGVA in a region that is conserved among anelloviruses. Two main clades, A and B, were distinguished in the phylogenetic tree (Fig. 2
). Branch B includes all of the clones with the shortest sequences, as well as several clones of 73 bp. Considering that the UTR region is well-conserved among anelloviruses, more differences are expected in comparison of the full-length genomes.
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Intra-organ variability was further evidenced by the sequences of 10 clones that were obtained from the lung of an animal from herd w. Six were strictly identical and grouped in branch B, whereas the other four, almost identical to one another, grouped in branch A, sharing 93 % identity with the six sequences of branch B (Fig. 2).
The lowest level of identity among our isolates was reached when comparing clones from two herds separated by 150 km (n and j) (Table 1). However, this level (78 %) was in the same range as the lowest level reached by two clones originating from the same herd, e.g. in herd i (81 %). This suggests that, although limited in number, our samples represent the global viral diversity in the region quite well.
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Based on a short sequence from a conserved domain, we showed that our sequences are related genetically to Sd-TTV31 and other Japanese isolates, with at least two subpopulations that are not restricted geographically, but mixed. Such a distribution suggests a common pool of genomes disseminated in the two regions, via an as-yet-unknown route. Hypothetically, dissemination could be amplified within an herd or a region by air-borne transmission, considering that the lung is the most frequently positive organ among those tested. The lung is also a site of virus accumulation in human beings (Okamoto et al., 2001a).
Whether swine anelloviruses represent a sanitary risk to their natural host or other hosts remains unknown. In our survey, most animals were healthy, although they were carriers of PCV2, supporting the hypothesis that the anellovirus isolates are not pathogenic in swine. However, the situation of anelloviruses in swine is reminiscent of the high worldwide prevalence of PCV2. This virus, which has coexisted with swine for a long time, has been associated with a recently emerged pathology, post-weaning multisystemic wasting syndrome, probably as a consequence of an as-yet-unidentified biological factor or a modern breeding practice. It appears that, in the same samples from Brittany, the diversity of anelloviruses is superior to that of PCV2, which exhibits a high degree of conservation in Brittany (de Boisséson et al., 2004). Despite the high host specificity demonstrated by anelloviruses to date and their non- or poor viability in heterologous hosts (Mushahwar et al., 1999
; Okamoto et al., 2000
), frequent exposure of human beings to a potentially heterogeneous inoculum produced by herds of numerous virus carriers is a sanitary risk that must not be neglected.
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ACKNOWLEDGEMENTS |
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Received 1 September 2004;
accepted 15 November 2004.