Molecular characterization of a novel astrovirus associated with disease in mink

Christian Mittelholzer1,{dagger}, Kjell-Olof Hedlund2, Lena Englund3, Hans-Henrik Dietz4 and Lennart Svensson1,{ddagger}

1 Department of Virology, Swedish Institute for Infectious Disease Control, S-171 82 Solna, Sweden
2 Department of Molecular Epidemiology and Biotechnology, Swedish Institute for Infectious Disease Control, S-171 82 Solna, Sweden
3 Department of Small Animals, National Veterinary Institute, S-751 89 Uppsala, Sweden
4 Department of Poultry, Fish and Fur Animals, Danish Veterinary Institute, DK-8200 Århus, Denmark

Correspondence
Christian Mittelholzer
christian.mittelholzer{at}imr.no


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Pre-weaning diarrhoea is a well-known problem in mink farming in Europe, causing morbidity that varies between farms, regions and season. Different causalities for the disease have been proposed, but only most recently has a novel astrovirus been identified as an important risk factor. In this report, the molecular characterization, origin and evolution of this novel astrovirus of mink are discussed. The polyadenylated, positive-stranded RNA genome was sequenced and found to contain 6610 nt, organized into three ORFs and two short UTRs. A ribosomal frameshift sequence links the 5' two ORFs, containing sequence motifs for a serine protease (ORF1a) and an RNA-dependent RNA polymerase (ORF1b). The structural proteins are encoded by ORF2 and, presumably, are expressed as a polyprotein precursor to be cleaved into the mature capsid proteins. These results indicate that mink astrovirus (MiAstV) has all of the features typical of members of the Astroviridae. Phylogenetic analyses revealed that MiAstV is distantly related to established astroviruses, showing less than 67 % similarity at the nucleotide level with its closest relative, ovine astrovirus, and even lower identities at the predicted amino acid level. Nevertheless, sequence analysis of MiAstV isolates from geographically distinct Swedish and Danish farms showed much less diversity. This suggests either the spread in the mink population of a virus that has evolved a long time ago or the recent introduction of an ancient virus into a new host species.

The GenBank accession number of the sequence reported in this paper is AY179509.

{dagger}Present address: Institute of Marine Research, Department of Aquaculture, N-5392 Storebø, Norway.

{ddagger}Present address: Department of Molecular and Clinical Medicine, University of Linköping, S-581 85 Linköping, Sweden.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
For several decades, a pre-weaning diarrhoea syndrome with unknown aetiology has been observed in mink farms in Europe. Numerous investigations studying the causality of this clinical syndrome have been conducted and various agents have been discussed as possible causes, but only recently did a case-controlled study reveal the presence of an astrovirus as a significant risk factor (Englund et al., 2002).

Astroviruses were named by Madeley & Cosgrove (1975) after their star-like appearance in electron microscopy; the name astrovirus comes from ‘astron’, which means star in Greek. Astroviruses are an important cause of acute infantile gastroenteritis in humans (Madeley & Cosgrove, 1975) and have been shown to infect sheep (Snodgrass & Gray, 1977), cattle (Woode & Bridger, 1978), dog (Williams, 1980), domestic cat (Hoshino et al., 1981), red deer (Tzipori et al., 1981), duck (Gough et al., 1984), mouse (Kjeldsberg & Hem, 1985), turkey (McNulty et al., 1980) and pig (Shimizu et al., 1990), causing various syndromes, ranging from mild diarrhoea in lambs (Snodgrass et al., 1979) and poult enteritis and mortality syndrome in turkeys (Yu et al., 2000) to acute nephritis in chickens (Imada et al., 2000).

The genome of astroviruses consists of a positive-stranded, polyadenylated RNA, which is about 7 kb long and contains three ORFs, designated ORF1a, ORF1b and ORF2 (Monroe et al., 1995). Whereas the latter encodes the capsid protein(s), ORF1a and 1b encode the nonstructural proteins involved in replication. All astrovirus genomes show a rather conserved frameshift slippery sequence between ORF1a and 1b (Jiang et al., 1993; Marczinke et al., 1994), ensuring that the latter is only translated as a fusion polyprotein together with ORF1a, with an efficiency of 25–28 % when compared to the translation of ORF1a alone (Lewis & Matsui, 1996). The products from all three ORFs are posttranslationally cleaved into mature proteins (Bass & Qiu, 2000; Geigenmüller et al., 2002a; Gibson et al., 1998; Kiang & Matsui, 2002; Willcocks et al., 1999). However, the above information is derived mainly from studies with human astroviruses and similar data about animal astroviruses are still scarce.

Animal and human astroviruses are grouped into the family Astroviridae (Matsui & Greenberg, 2001) due to the similarity in genome organization but, in general, they lack regions of high nucleotide sequence identity. Nevertheless, conserved amino acid motifs, such as a serine protease motif or features typical of RNA-dependent RNA polymerases (RdRp), have been identified in the genomes of all astroviruses (Jiang et al., 1993). Interestingly, a conserved nuclear localization signal has also been identified in the astrovirus genome, characterized by two clusters of basic amino acid residues separated by a spacer region of 10 aa (Dingwall & Laskey, 1991). Observations made with bovine astrovirus (Aroonprasert et al., 1989) and recent reports of human astrovirus type 1 (HAstV-1) indicate that this signal is indeed functional, directing ORF1a products to the nucleus of infected cells in vitro (Willcocks et al., 1999). Another region showing at least some conservation among human strains and between human and animal astroviruses is the N-terminal part of the ORF2-encoded polyprotein, presumably forming the capsid protein domain that maintains the interaction with the genomic RNA (Mendez et al., 2002; Geigenmüller et al., 2002b).

In this study, we report the molecular characterization of mink astrovirus (MiAstV). The genomic sequence was determined and revealed features typical of members of the family Astroviridae. While comparison of the nucleotide and deduced amino acid sequences showed that MiAstV is distantly related to other astroviruses, only limited genetic variability was observed among a number of Danish and Swedish isolates. This indicates that either an ancient virus entered into a new host species or that the virus has evolved a long time ago and is endemic in the mink population.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mink samples.
The sampling of mink kits has been described in a previous report (Englund et al., 2002). Briefly, intestinal and faecal samples were collected from 180 mink kits, sacrificed in eight Swedish and ten Danish mink farms. Each sample was placed in a vial with 2 ml PBS, containing 1 % penicillin/streptomycin and 1 % fungizone. Samplings were done as a case-control study, where 50 % of the samples represent healthy kits in problem-free farms (control farms), 25 % of the samples represent clinically healthy kits in farms affected by pre-weaning diarrhoea (case farms) and 25 % of the samples represent affected kits in case farms.

RNA extraction.
Total RNA was extracted with Trizol (Gibco-BRL), according to the manufacturer's instructions. Briefly, 250 µl of intestinal/faecal sample was mixed thoroughly with 750 µl Trizol. Subsequently, 200 µl chloroform was added and the tube was shaken vigorously by hand. After a 5 min period of incubation, the tube was centrifuged at 12 000 g for 15 min and 600 µl of the upper phase was transferred to a fresh 1·5 ml tube. Total RNA was precipitated with 500 µl isopropanol overnight at -20 °C and then pelleted at 12 000 g for 30 min. The pellet was washed twice with 70 % ethanol, dried and dissolved in 20 µl DEPC-treated water. Extracted RNA was either used directly or stored at -20 °C until use.

Primers and amplification strategy.
Based on an alignment of full-length sequences from human and animal astroviruses [ANV, avian nephritis virus (AB033998); HAstV-3 (AF141381); TAstV-2, turkey astrovirus type 2 (AF206663); HAstV-8 (AF260508); HAstV-2 (L13745); HAstV-1a (L23513); HAstV-1b (NC_001943); TAstV-1 (Y15936); OAstV, ovine astrovirus (Y15937)], primers were designed to amplify a short stretch of the conserved region within the RdRp-encoding sequence. Primer MA2 was used for reverse transcription and primers MA2 and MA4 were employed for PCR. All PCR and sequencing primer sequences are shown in Table 1. New primers were designed from the two sequences obtained in the first amplification. The 3' part of the genome was amplified by 5'RACE using the GeneRacer kit (Invitrogen), employing the antisense primer GeneRacer3' and MA7 as sense primer. The sequence between the RdRp and protease regions was amplified using primers MA8 (antisense) and MA9 (sense). To amplify more of the 5' part of the genome, PCR was performed with primers MA10 (antisense) and random hexamers (sense). The 5' most sequence was obtained by 5'RACE using primer MA28 for reverse transcription and first-round PCR and primers Not-T7 and GeneRacer5'nested as first- and second-round PCR primers, respectively. Fig. 1 shows a schematic overview of the amplification strategy and the sequencing process.


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Table 1. Primer sequences used in this study

Primers used for PCR, antisense (–) or sense (+), and sequencing (S) are shown.

 


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Fig. 1. (a) A schematic overview of the MiAstV genome. Grey bars represent the three ORFs. Open bars represent specific protein motifs. Prot, Protease domain; NLS, nuclear localization signal; RdRp, RNA-dependent RNA polymerase domain. (b) The four large PCR fragments are shown as black bars, together with their name and approximate size. The primers used for PCR amplification and sequencing are shown as arrows.

 
Reverse transcription.
Extracted RNA (10 µl) was reverse-transcribed at 37 °C for 1 h after adding 10 µl reverse transcription mixture containing 50 pmol antisense primer, 200 units Superscript II (Gibco-BRL), 1x first-strand buffer, 10 mM DTT and 10 mM each dNTP (Pharmacia). When using the GeneRacer kit, the manufacturer's instructions were followed. Briefly, total extracted RNA was dephosphorylated with calf intestinal phosphatase, decapped with tobacco acid pyrophosphatase and ligated to the GeneRacer RNA oligonucleotide. The ligated RNA was then reverse-transcribed with AMV reverse transcriptase and either the oligo(dT) primer (3' end amplification and internal fragments) or the MiAstV-specific primer MA28 (5' end amplification).

PCR and cloning.
Amplifications of the short fragments in the conserved protease and RdRp regions were performed in a DNA Thermal Cycler (Perkin-Elmer) using Taq DNA polymerase (AmpliTaq, Perkin-Elmer). The reaction mixtures comprised 1x PCR buffer, 2·5 mM MgCl2, 200 µM each dNTP, 15 pmol primers MA2 and MA4, 2·5 units Taq DNA polymerase and 5 µl cDNA. Amplification involved a 2 min denaturation step at 94 °C, 5 cycles of denaturation at 94 °C for 30 s, primer annealing at 55 °C for 30 s and primer extension at 72 °C for 1 min, followed by 5 cycles at an annealing temperature of 53 °C and 20 cycles at 51 °C. After a final extension step of 7 min at 72 °C, 10 µl of the reactions were run on 2 % agarose gels and the DNA bands were visualized under UV light after ethidium bromide staining.

When amplifying the larger fragments in the middle and at the 3' end of the genome, ThermoZyme (a polymerase mixture with proofreading activity) was used. Briefly, 1 µl cDNA was amplified in a 50 µl reaction containing 10 µl 5x buffer, 1 µl 10 mM each dNTP, 15 pmol each primer and 1 µl ThermoZyme. After an initial denaturation, the PCR profile was 5 cycles of denaturation at 94 °C for 30 s and primer extension at 72 °C for 7 min, 5 cycles of denaturation at 94 °C for 30 s, primer annealing at 70 °C for 30 s and primer extension at 72 °C for 3 min, followed by 20 cycles of denaturation at 94 °C for 30 s, primer annealing at 68 °C for 30 s and primer extension at 72 °C for 3 min. The ends of the amplicons were polished by a final extension step for 10 min at 72 °C. PCR products were analysed as described above.

To amplify the remaining 5' end sequence, a two-step strategy was applied. In first-round PCR, the MiAstV-specific reverse primer MA10 was used in combination with random hexamers (Pharmacia) as sense primers, following essentially the PCR profile described for the other ThermoZyme PCRs. This did not result in the amplification of the very 5' end of the viral genome. Therefore, in a second step to amplify the 5' most sequence, we ligated the anchor oligonucleotide T7-Not to the 3' end of the cDNA to allow for a nested, anchor–ligation PCR strategy to be performed. Subsequently, oligonucleotide Not-T7 (complementary to the ligated anchor oligonucleotide) and the MiAstV-specific primer MA26 were used in a PCR profile comprising an initial denaturation for 5 min, 40 cycles of denaturation at 94 °C for 30 s and annealing/extension at 72 °C for 90 s and a final extension step at 72 °C for 7 min. In the second, nested round of PCR, the GeneRacer5'nested primer and the MiAstV-specific primer MA28 were used. The PCR profile consisted of 5 cycles of denaturation at 94 °C for 30 s and primer extension at 72 °C for 1 min, 5 cycles of denaturation at 94 °C for 30 s, primer annealing at 65 °C for 30 s and primer extension at 72 °C for 1 min, followed by 20 cycles of denaturation at 94 °C for 30 s, primer annealing at 60 °C for 30 s and primer extension at 72 °C for 1 min.

For cloning, the remaining PCRs were run on preparative agarose gels, purified using the QIAEX II kit (Qiagen), following the manufacturer's instructions, and cloned into a plasmid vector using the TOPO-TA cloning kit (Invitrogen). Bacterial colonies were screened by PCR for the presence of the expected insert using the T3 and T7 primers provided in the cloning kit.

Sequence determination.
Cycle sequencing was performed on plasmid DNA preparations with the BigDye Terminator Cycle Sequencing kit (Perkin-Elmer) on an automated ABI PRISM Model 3100 sequencer. After initial sequencing using the plasmid-specific T3 and T7 primers, MiAstV-specific primers were designed (see Table 1) and overlapping fragments from at least two clones were sequenced. In the case of the 13 ambiguities, a third clone was sequenced and the most common nucleotide incorporated into the consensus sequence. In the case of the shorter fragments, both strands of one clone were sequenced using the T3 and T7 primers. Nucleotide sequences, all derived from a single sample and therefore most probably representing a single virus strain, were assembled and analysed. Amino acid sequences were predicted using the programs of DNASTAR.

Phylogeny.
Phylogenetic analyses were performed using the PHYLIP package (version 3.573). The BIOEDIT software was used to manipulate the sequences retrieved from GenBank. As the PHYLIP program does not accept sequences of variable length, all sequences had to be imported first into BIOEDIT. Sequences were aligned using the CLUSTAL_W algorithm and then the sequences containing gaps in regions of low similarity were exported for use in PHYLIP. A phylogenetic tree without bootstrapping was calculated using the DNADIST (kimura's 2-parameter distance estimation) and NEIGHBOR (neighbour-joining method) programs and displayed by DRAWTREE (all programs of the PHYLIP package). This results in branches that represent actual distances between genomic sequences (since all information available was used to build the tree). Bootstrap resampling (where only a subset of all information is used in any given round) and subsequent construction of a consensus tree from 1000 datasets (CONSENSE) was performed to test the robustness of the phylogenetic comparison. The information obtained shows how often a certain branch shows up despite the lack of a part of the data each time when an individual tree was constructed. Only bootstrap values above 70 per cent are considered significant.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sequence determination
Alignment of full-length genomic sequences of human and animal astroviruses available in GenBank showed considerable variability. Nevertheless, we identified a stretch of conserved amino acids and nucleotide residues in the RdRp region. There was another rather conserved nucleotide sequence about 400 nt upstream of this site. This allowed us to design the slightly degenerate primers MA2 and MA4 (see Table 1), which should be able to amplify a stretch of about 400 nt from all human or animal astroviruses, including the novel astrovirus found in mink. When RT-PCR using these primers was performed on two mink samples showing large amounts of virus by electron microscopy (one from a Danish mink farm and one from a Swedish mink farm), amplicons of the expected size were obtained. To our surprise, cloning and sequencing of several clones representing these PCR fragments revealed two very different sequences. Whereas processing of the Danish mink sample resulted in the amplification of the expected RdRp-like sequence, the same primers obviously had amplified a sequence in the protease region from the Swedish mink sample. Knowledge about these two parts of the genome, which were about 2 kb apart, together with the presumed presence of a poly(A) tail in analogy to all known astroviruses, enabled us to amplify the 3' two-thirds of the MiAstV genome from the Swedish mink sample. Applying primers GeneRacer3', MA7, MA8 and MA9 resulted in two large PCR fragments of about 3 and 2 kb, respectively (Fig. 1). A primer walking strategy using various primers listed in Table 1 was then applied to determine the sequence of these amplicons.

Only a limited amount of material from the 3- to 6-week-old mink kits was available. Therefore, we decided to try unconventional amplification approaches to determine the 5' remaining sequences of MiAstV. Since the previous sequences had been determined from one single sample from a Swedish farm, we wanted to complete the genomic sequence from the same source of material. Therefore, amplification using random hexamers, normally used only for random priming during reverse transcription, was attempted and resulted in the production of amplicons covering about two-thirds of the missing 5' sequences. The 5' most sequence was obtained by a nested, anchor–ligation PCR strategy and sequencing several clones from two independent PCRs ensured that the 5' end was determined accurately. Two clones showed a 5' deletion of 35 nt compared to the other four clones, which all showed the same sequence. Since the latter were derived from two independent PCR runs, we concluded that these sequences represent the accurate 5' end of MiAstV.

Genomic analysis
The positive-stranded RNA genome was shown to be polyadenylated and contained 6610 nt upstream of a poly(A) tail of undetermined length. The three clones sequenced at the very 3' end showed 23, 48 and 55 adenosine residues, respectively, between the MiAstV unique sequence and the cloning vector sequence. These numbers are presumably lower than the true number of A residues in the actual poly(A) tail due to the amplification technique using a poly(dT) primer.

Sequence analysis revealed that the unique sequence was organized into three ORFs of 2652 (ORF1a), 1557 (ORF1b) and 2328 nt (ORF2), a short 5' UTR of 26 nt and a 3' UTR of 108 nt. Similarity searches with parts of the nucleotide sequence or the complete sequence using the BLAST search engine resulted initially in only a few hits, with similarities over a considerable sequence length. However, when searches were performed with deduced amino acid sequences from each of the ORFs, more and longer sequence stretches showing similarity to the query sequence were obtained; the hits with the highest similarity scores consisted exclusively of astrovirus sequences. This demonstrated that this novel virus was indeed a member of the family Astroviridae and all further comparisons were done using astrovirus sequences available in GenBank.

In pairwise comparisons using the BLAST2 program, OAstV was found to be the closest relative to MiAstV, showing amino acid identities of 63, 49 and 45 % for ORF1a, ORF1b and ORF2, respectively. Similar results were obtained when the MEGALIGN program of the DNASTAR package was employed to construct phylogenetic trees with all available astrovirus sequences for each ORF. These analyses confirmed further the distant relationship of MiAstV with the other astroviruses and OAstV as being the closest relative.

Analysis of individual ORFs
To confirm further the identity of MiAstV as an astrovirus and to assess the integrity of the sequence determined, the genomic nucleotide sequence and the amino acid sequence deduced separately for each of the three ORFs were searched using a variety of nucleotide and protein analysis programs. By analogy to other astroviruses, a ribosomal frameshift sequence (Jiang et al., 1993; Marczinke et al., 1994) was observed in the overlapping region between ORF1a and 1b. The heptameric AAAAAAC sequence at positions 2615–2621 is followed by a stem–loop sequence at positions 2632–2644. The conserved heptameric sequence motif has been shown to be an absolute requirement, whereas the stem–loop region seems to be dispensable for the frameshift mechanism (Lewis & Matsui, 1996). This feature allows the expression of ORF1b and ensures that the proteins encoded by this ORF are only translated as a fusion polyprotein together with ORF1a, with an efficiency of 25–28 % when compared to the translation of ORF1a alone (Lewis & Matsui, 1996).

Comparisons with deduced amino acid sequences for other astroviruses for which full-length sequences were available revealed the presence of a number of characteristic amino acid motifs. In ORF1a, a serine protease domain was predicted and detailed sequence alignment with corresponding regions from other astroviruses showed that all catalytically active residues as well as a number of other amino acids were conserved between all astroviruses (Fig. 2). In addition, five possible transmembrane domains could be located in the N-terminal half of ORF1a (Fig. 3) by the TMHMM program (Krogh et al., 2001; http://www.cbs.dtu.dk/services/TMHMM-2·0/). When analysing further the deduced amino acid sequence for ORF1a, a bipartite nuclear localization signal was also identified. It is characterized by two clusters of basic amino acid residues separated by a spacer region of 10 aa (Dingwall & Laskey, 1991). Starting slightly upstream of the actual frameshift slippery sequence described above, ORF1b is the shortest of the three large coding regions detected in the MiAstV genome. It harbours amino acid motifs of an RdRp that is homologous to members of the superfamily I or picornavirus-like supergroup of RNA polymerases. However, it does not contain an obvious or known helicase motif. The presumed capsid protein(s) are encoded by ORF2 situated in the 3' part of the genome. No internal ribosomal entry site preceding this ORF was found and it can, therefore, be assumed that it is transcribed from the genome as a subgenomic RNA (Monroe et al., 1993). The N-terminal region of the MiAstV ORF2 shows a higher degree of similarity than the C-terminal half of the predicted protein when compared to other astrovirus capsid protein sequences.



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Fig. 2. Alignment of the putative serine protease. The predicted amino acid sequences of the serine protease from ORF1a of full-length astrovirus genomes were aligned. Residues in bold are completely conserved in all 10 sequences. The suspected catalytic triad is underlined. Numbers before and after the sequences are the first and last positions of the sequences shown, counted from the first amino acid of ORF1a.

 


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Fig. 3. Predicted transmembrane domains in ORF1a. The predicted amino acid sequence of the MiAstV ORF1a was subjected to the TMHMM program. Putative transmembrane domains and their probability are indicated by grey areas and their size, respectively. The top panel shows a schematic overview with the five transmembrane domains (squares) and the predicted localization of the hydrophilic parts of the protein inside (dark grey lines) or outside (light grey lines) the membrane, respectively. The x-axis shows the numbers of amino acids.

 
Phylogeny
The full-length genomic sequence of MiAstV was phylogenetically compared to the nine complete genomic sequences for astroviruses available in GenBank. The resulting tree (Fig. 4) shows clearly that MiAstV is distantly related to other astroviruses but most closely related to OAstV. The high bootstrapping numbers at branching points between the astrovirus species show that the tree was indeed very robust, with only the very homogeneous group of the human astroviruses showing a less clear distinction capability. Also visible in the tree is the boundary between the genus Mamastrovirus, encompassing all mammalian astroviruses, and the genus Avastrovirus, comprising the avian astroviruses, which are newly established to replace the previously unique genus Astrovirus (Mayo, 2002).



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Fig. 4. Phylogenetic analysis of complete astrovirus genomes. A phylogenetic tree was constructed using the DNADIST and NEIGHBOR programs of the PHYLIP package. Bootstrapping numbers are expressed as per cent of 1000 replicates. The dashed line indicates the border between the genus Avastrovirus (upper part) and the genus Mamastrovirus (lower part). The closely related human astroviruses are shown in the inset. ANV, Avian nephritis virus (AB033998); TAstV-1, turkey astrovirus type 1 (Y15936); TAstV-2, turkey astrovirus type 2 (AF206663); MiAstV, mink astrovirus (AY179509); OAstV, ovine astrovirus (Y15937); HAstV-1a, human astrovirus type 1 (L23513); HAstV-1b, human astrovirus type 1 (NC_001943); HAstV-2, human astrovirus type 2 (L13745); HAstV-3, human astrovirus type 3 (AF141381); HAstV-8, human astrovirus type 8 (AF260508).

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Many potential causes of pre-weaning diarrhoea in mink have been presented since this disease was described in the 1950s (anecdotal evidence). In a case-controlled epidemiological study, we could establish a strong link between the presence of astrovirus particles in mink faeces and disease problems in farms (Englund et al., 2002). Since this was also true on the level of individual animals, we are confident to have identified a cause of the disease, or at least identified an important factor contributing to its aetiology. To prove the nature of the particles observed by electron microscopy and to molecularly characterize the virus, the full-length genomic sequence of this novel virus was determined in this study.

Despite extensive sequence diversity among astroviruses (Lukashov & Goudsmit, 2002), initial sequence data were readily obtained using the applied strategy. Surprisingly, using a single primer pair, we obtained an amplicon of the expected size for the region that was targeted, and also a region situated about 2 kb upstream in the astrovirus genome. This demonstrated the suitability of the chosen amplification protocol. Interestingly, we could only identify the second sequence as being derived from an astrovirus when we performed BLAST searches with the deduced amino acid sequence, indicating that sequence identities between astroviruses of different species are more prominent on the protein level than on the genomic level.

As for other emerging viruses (Bowden et al., 2001; Haqshenas et al., 2001; Smiley et al., 2002; Todd et al., 2001), a range of amplification strategies designed for the determination of unknown sequences had to be applied to obtain the complete genome. It was important to perform this work using a single sample, despite limits of material, in order to avoid differences due to the possible sequence diversity among astrovirus strains present in the different samples. It was, therefore, of great help that we initially obtained two sequences located about 2 kb apart, facilitating the rapid amplification and sequence determination of the 3' two-thirds of the genome. The presence of poly(A) tails in all 3' most sequence parts indicated that the genome is polyadenylated, as is the case for all other astroviruses for which full-length sequences have been determined to date (Imada et al., 2000; Jiang et al., 1993; Koci et al., 2000; Lewis et al., 1994; Mendez-Toss et al., 2000; Oh & Schreier, 2001; Willcocks et al., 1994). The 5' most extreme sequence proved to be more difficult to obtain. However, different approaches resulted in the independent observation of only two types of cloned inserts. The sequences obtained from these clones differed from each other only by the presence of a 35 nt deletion in some of the clones. Since both sequence variants were present in all amplifications, we believe that the longer sequence represents the true genomic sequence. The slightly shorter products might be due to the presence of strong secondary structures. The strategy to sequence at least two clones of each part was justified due to the presence of differing nucleotides at some positions. Nevertheless, these occasions were rare and sequencing of a third clone always resulted in the unambiguous assignment of a certain base at a particular position. Together with the known high fidelity of the DNA polymerases used, this indicates that the virus population in the single sample used was rather homogeneous and that the consensus sequence obtained represents the whole genome of a mink astrovirus.

Performing similarity or BLAST searches with the nucleotide sequences turned out to be a real challenge. Low identity with other sequences in the databases resulted in only a few hits or only short stretches of considerable similarity, even though conserved parts were contained in the query sequences. However, the genomic structure and the amino acid sequence motifs identified in the three ORFs clearly showed that it is an astrovirus.

The polyadenylated, positive-stranded RNA of 6610 nt is organized into three ORFs of 2652, 1557 and 2328 nt, and two short UTRs of 26 and 108 nt at the 5' and 3' ends, respectively. The ribosomal frameshift sequence linking ORF1a and 1b is a feature typical of astroviruses (Lewis & Matsui, 1995, 1996, 1997; Marczinke et al., 1994), as are the amino acid motifs for a serine protease in ORF1a and the RdRp of the picornavirus supergroup lacking a helicase motif in ORF1b. Comparisons of the deduced amino acid sequences from ORF1a, 1b and 2 of MiAstV with the corresponding ORFs of its closest relative, OAstV, revealed identities of 38, 63 and 37 %, respectively. When the same was done using the nucleotide sequences, the numbers were quite different for ORF1a (55 %) and ORF2 (30 %), with the exception of ORF1b, which showed 64 % identity also on the nucleotide level. This might indicate that the evolutionary clock for the three genes and the proteins encoded by them might be different or that sequence or structural constraints are limiting the variability for some but not all ORFs of the astrovirus genome. Interestingly, the 5' half of ORF2 showed much higher similarity than the 3' part, especially on the amino acid level. This probably results from the presumed interaction of the N-terminal part of the capsid protein with the genomic RNA (Mendez et al., 2002; Geigenmüller et al., 2002b). Nevertheless, more functional studies using single mature proteins and a larger number of full-length genomes are required to shed more light on these issues.

We conclude from the sequence analysis of the MiAstV genome that this novel virus is most probably specific for mink, either farmed or wild. It is tempting to speculate either that this virus originates from free-ranging animals from closely related carnivore species and repeatedly crosses the species barrier or that it circulates constantly in the farmed mink population, only causing disease in young mink kits when they are born during a relatively short period of time.


   ACKNOWLEDGEMENTS
 
This work was supported by research grants received from the Danish Fur Breeders Research Centre.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Aroonprasert, D., Fagerland, J. A., Kelso, N. E., Zheng, S. & Woode, G. N. (1989). Cultivation and partial characterization of bovine astrovirus. Vet Microbiol 19, 113–125.[CrossRef][Medline]

Bass, D. M. & Qiu, S. (2000). Proteolytic processing of the astrovirus capsid. J Virol 74, 1810–1814.[Abstract/Free Full Text]

Bowden, T. R., Westenberg, M., Wang, L. F., Eaton, B. T. & Boyle, D. B. (2001). Molecular characterization of Menangle virus, a novel paramyxovirus which infects pigs, fruit bats, and humans. Virology 283, 358–373.[CrossRef][Medline]

Dingwall, C. & Laskey, R. A. (1991). Nuclear targeting sequences: a consensus? Trends Biochem Sci 16, 478–481.[CrossRef][Medline]

Englund, L., Chriél, M., Dietz, H. H. & Hedlund, K. O. (2002). Astrovirus epidemiologically linked to pre-weaning diarrhoea in mink. Vet Microbiol 85, 1–11.[CrossRef][Medline]

Geigenmüller, U., Chew, T., Ginzton, N. & Matsui, S. M. (2002a). Processing of nonstructural protein 1a of human astrovirus. J Virol 76, 2003–2008.[Abstract/Free Full Text]

Geigenmüller, U., Ginzton, N. H. & Matsui, S. M. (2002b). Studies on intracellular processing of the capsid protein of human astrovirus serotype 1 in infected cells. J Gen Virol 83, 1691–1695.[Abstract/Free Full Text]

Gibson, C. A., Chen, J., Monroe, S. A. & Denison, M. R. (1998). Expression and processing of nonstructural proteins of the human astroviruses. Adv Exp Med Biol 440, 387–391.[Medline]

Gough, R. E., Collins, M. S., Borland, E. & Keymer, L. F. (1984). Astrovirus-like particles associated with hepatitis in ducklings. Vet Rec 114, 279.

Haqshenas, G., Shivaprasad, H. L., Woolcock, P. R., Read, D. H. & Meng, X. J. (2001). Genetic identification and characterization of a novel virus related to human hepatitis E virus from chickens with hepatitis–splenomegaly syndrome in the United States. J Gen Virol 82, 2449–2462.[Abstract/Free Full Text]

Hoshino, Y., Zimmer, J. F., Moise, N. S. & Scott, F. W. (1981). Detection of astroviruses in feces of a cat with diarrhea. Arch Virol 70, 373–376.[Medline]

Imada, T., Yamaguchi, S., Mase, M., Tsukamoto, K., Kubo, M. & Morooka, A. (2000). Avian nephritis virus (ANV) as a new member of the family Astroviridae and construction of infectious ANV cDNA. J Virol 74, 8487–8493.[Abstract/Free Full Text]

Jiang, B., Monroe, S. S., Koonin, E. V., Stine, S. E. & Glass, R. I. (1993). RNA sequence of astrovirus: distinctive genomic organization and a putative retrovirus-like ribosomal frameshifting signal that directs the viral replicase synthesis. Proc Natl Acad Sci U S A 90, 10539–10543.[Abstract]

Kiang, D. & Matsui, S. M. (2002). Proteolytic processing of a human astrovirus nonstructural protein. J Gen Virol 83, 25–34.[Abstract/Free Full Text]

Kjeldsberg, E. & Hem, A. (1985). Detection of astroviruses in gut contents of nude and normal mice. Arch Virol 84, 135–140.[Medline]

Koci, M. D., Seal, B. S. & Schultz-Cherry, S. (2000). Molecular characterization of an avian astrovirus. J Virol 74, 6173–6177.[Abstract/Free Full Text]

Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. (2001). Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305, 567–580.[CrossRef][Medline]

Lewis, T. L. & Matsui, S. M. (1995). An astrovirus frameshift signal induces ribosomal frameshifting in vitro. Arch Virol 140, 1127–1135.[Medline]

Lewis, T. L. & Matsui, S. M. (1996). Astrovirus ribosomal frameshifting in an infection-transfection transient expression system. J Virol 70, 2869–2875.[Abstract]

Lewis, T. L. & Matsui, S. M. (1997). Studies of the astrovirus signal that induces (-1) ribosomal frameshifting. Adv Exp Med Biol 412, 323–330.[Medline]

Lewis, T. L., Greenberg, H. B., Herrmann, J. E., Smith, L. S. & Matsui, S. M. (1994). Analysis of astrovirus serotype 1 RNA, identification of the viral RNA-dependent RNA polymerase motif, and expression of a viral structural protein. J Virol 68, 77–83.[Abstract]

Lukashov, V. V. & Goudsmit, J. (2002). Evolutionary relationships among Astroviridae. J Gen Virol 83, 1397–1405.[Abstract/Free Full Text]

Madeley, C. R. & Cosgrove, B. P. (1975). 28 nm particles in faeces in infantile gastroenteritis. Lancet ii, 451–452.

Marczinke, B., Bloys, A. J., Brown, T. D., Willcocks, M. M., Carter, M. J. & Brierley, I. (1994). The human astrovirus RNA-dependent RNA polymerase coding region is expressed by ribosomal frameshifting. J Virol 68, 5588–5595.[Abstract]

Matsui, S. M. & Greenberg, H. B. (2001). Astroviruses. In Fields Virology, 4th edn, pp. 875–893. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.

Mayo, M. A. (2002). Virology division news: ICTV at the Paris ICV: Results of the Plenary Session and the Binomial Ballot. Arch Virol 147, 2254–2260.[CrossRef]

McNulty, M. S., Curran, W. L. & McFerran, J. B. (1980). Detection of astroviruses in turkey faeces by direct electron microscopy. Vet Rec 106, 561.[Medline]

Mendez, E., Fernandez-Luna, T., Lopez, S., Mendez-Toss, M. & Arias, C. F. (2002). Proteolytic processing of a serotype 8 human astrovirus ORF2 polyprotein. J Virol 76, 7996–8002.[Abstract/Free Full Text]

Mendez-Toss, M., Romero-Guido, P., Munguia, M. E., Mendez, E. & Arias, C. F. (2000). Molecular analysis of a serotype 8 human astrovirus genome. J Gen Virol 81, 2891–2897.[Abstract/Free Full Text]

Monroe, S. S., Jiang, B., Stine, S. E., Koopmans, M. & Glass, R. I. (1993). Subgenomic RNA sequence of human astrovirus supports classification of Astroviridae as a new family of RNA viruses. J Virol 67, 3611–3614.[Abstract]

Monroe, S. S., Carter, M. J., Herrmann, J. E., Kurtz, J. B. & Matsui, S. M. (1995). Family Astroviridae. In Virus Taxonomy. Sixth Report of the International Committee on Taxonomy of Viruses, pp. 364–367. Edited by F. A. Murphy, C. M. Fauquet, D. H. L. Bishop, S. A. Ghabrial, A. W. Jarvis, G. P. Martelli, M. A. Mayo & M. D. Summers. Vienna & New York: Springer-Verlag.

Oh, D. & Schreier, E. (2001). Molecular characterization of human astroviruses in Germany. Arch Virol 146, 443–455.[CrossRef][Medline]

Shimizu, M., Shirai, J., Narita, M. & Yamane, T. (1990). Cytopathic astrovirus isolated from porcine acute gastroenteritis in an established cell line derived from porcine embryonic kidney. J Clin Microbiol 28, 201–206.[Medline]

Smiley, J. R., Chang, K. O., Hayes, J., Vinje, J. & Saif, L. J. (2002). Characterization of an enteropathogenic bovine calicivirus representing a potentially new calicivirus genus. J Virol 76, 10089–10098.[Abstract/Free Full Text]

Snodgrass, D. R. & Gray, E. W. (1977). Detection and transmission of 30 nm virus particles (astroviruses) in faeces of lambs with diarrhoea. Arch Virol 55, 287–291.[Medline]

Snodgrass, D. R., Angus, K. W., Gray, E. W., Menzies, J. D. & Paul, G. (1979). Pathogenesis of diarrhoea caused by astrovirus infections in lambs. Arch Virol 60, 217–226.[Medline]

Todd, D., Weston, J. H., Soike, D. & Smyth, J. A. (2001). Genome sequence determinations and analyses of novel circoviruses from goose and pigeon. Virology 286, 354–362.[CrossRef][Medline]

Tzipori, S., Menzies, J. D. & Gray, E. W. (1981). Detection of astrovirus in the faeces of red deer. Vet Rec 108, 286.[Medline]

Willcocks, M. M., Brown, T. D., Madeley, C. R. & Carter, M. J. (1994). The complete sequence of a human astrovirus. J Gen Virol 75, 1785–1788.[Abstract]

Willcocks, M. M., Boxall, A. S. & Carter, M. J. (1999). Processing and intracellular location of human astrovirus non-structural proteins. J Gen Virol 80, 2607–2611.[Abstract/Free Full Text]

Williams, F. P., Jr (1980). Astrovirus-like, coronavirus-like, and parvovirus-like particles detected in the diarrheal stools of beagle pups. Arch Virol 66, 215–226.[Medline]

Woode, G. N. & Bridger, J. C. (1978). Isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves. J Med Microbiol 11, 441–452.[Abstract]

Yu, M., Tang, Y., Guo, M., Zhang, Q. & Saif, Y. M. (2000). Characterization of a small round virus associated with the poult enteritis and mortality syndrome. Avian Dis 44, 600–610.[Medline]

Received 1 April 2003; accepted 9 July 2003.



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