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
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ABSTRACT |
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The GenBank accession number of the sequence reported in this paper is AY179509.
Present address: Institute of Marine Research, Department of Aquaculture, N-5392 Storebø, Norway.
Present address: Department of Molecular and Clinical Medicine, University of Linköping, S-581 85 Linköping, Sweden.
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INTRODUCTION |
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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 2528 % 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.
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METHODS |
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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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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, anchorligation 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.
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RESULTS |
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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, anchorligation 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 26152621 is followed by a stemloop sequence at positions 26322644. The conserved heptameric sequence motif has been shown to be an absolute requirement, whereas the stemloop 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 2528 % 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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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Received 1 April 2003;
accepted 9 July 2003.
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