1 Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands
2 Centre de Biophysique Moléculaire, CNRS, Orléans, France
3 GSF-Institute for Ecological Chemistry, D-85764 Oberschleissheim, Germany
Correspondence
Monique M. van Oers
monique.vanoers{at}wur.nl
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the nucleotide sequence determined in this work is AY251269.
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INTRODUCTION |
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The monocistronic viruses have been grouped in the genus Iflavirus (Christian et al., 2002), which has not yet been placed in a virus family. The type species of this genus is Infectious flacherie virus (IFV), which was isolated from the silkworm Bombyx mori (Isawa et al., 1998
). Two species have officially been assigned to this genus: Sacbrood virus (SBV) of bees (Ghosh et al., 1999
) and Perina nuda picorna-like virus (PnPV) (Wu et al., 2002
). Deformed wing virus (DWV) (GenBank accession nos AY292384 and AJ489744) of honey bees and the recently analysed Kakugo virus (KV) (Fujiyuki et al., 2004
), which was isolated from the brains of aggressive worker honey bees, are related closely to each other and have the characteristics of iflaviruses. Another potential member of the genus Iflavirus is Ectropis obliqua picorna-like virus (EoPV) (Wang et al., 2004
), which was isolated from the moth Ectropis obliqua. Iflaviruses have a single, large ORF that encodes both the structural and non-structural proteins. As with picornaviruses of vertebrates, the capsid proteins are encoded by the 5' region of the genome and the proteins involved in virus replication by the 3' region part. However, unlike vertebrate picornaviruses, iflaviruses lack the 2A-protease/2B/2C region (van Regenmortel et al., 2000
), as shown in Fig. 1
.
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In the search for pathogens of the mite Varroa destructor, we isolated virus particles with a mean diameter of 27 nm that resembled picorna-like viruses from mites in beehives at Wageningen University. Picorna-like virus particles have previously been observed in Varroa mites (Kleespies et al., 2000). The complete sequence of the viral genome was characterized and phylogenetic analysis was performed on the RNA-dependent RNA polymerase (RdRp) to determine the taxonomic position of this virus, tentatively named Varroa destructor virus 1 (VDV-1). To determine whether this virus replicates in the mites, a PCR test was developed to detect the complementary (negative) strand of the viral genome, which serves as a replication intermediate.
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METHODS |
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SDS-PAGE, Western blot analysis and N-terminal sequencing.
The structural proteins of the purified virus were resolved by 12 % SDS-PAGE and the proteins were blotted onto a PVDF membrane. The two large structural proteins, with molecular masses of approximately 46 and 32 kDa, were N-terminally sequenced at Centre de Biophysique Moléculaire, CNRS, France. Antibodies raised in rabbits against the purified virus after primary and booster injections were tested against the virus structural proteins in a Western blot analysis.
Electron microscopy of mite tissue.
Mites were suspended in distilled water and their appendages were removed. Their bodies were then fixed for 1·5 h at room temperature with 3 % paraformaldehyde in 0·1 M potassium phosphate buffer, pH 7·2, with 2·5 % (w/v) sucrose. After fixation, mites were washed three times with 0·1 M potassium phosphate buffer, pH 7·2, at 4 °C and kept overnight in the same buffer. Mites were dehydrated by using serial ethanol solutions as follows: 50 % ethanol at 0 °C for 30 min, 50 % ethanol at 20 °C for 30 min, 70 % ethanol at 20 °C for 30 min, 90 % ethanol at 20 °C for 45 min and 100 % ethanol at 20 °C for 60 min. Mites were transferred to a 100 % ethanol : LR Gold resin mixture (1 : 1) and kept at 20 °C for 1 h. Mites were then placed in pure LR Gold resin and kept overnight at 20 °C. Embedding of mites in LR Gold resin was done by UV-mediated polymerization of the resin for 48 h at 20 °C. Embedded mites were sliced into 60 nm sections and labelled for observation by immunogold with antibodies raised in rabbits using the purified virus sample.
RNA isolation.
RNA was isolated from 20 µl virus suspension (sample used for sequencing the virus genome) or approximately 50 live mites (sample used for detection of replication). Mite bodies were ground in the presence of RNasin (Promega) prior to extraction. RNA extraction was done by using 800 µl TRIzol reagent (Invitrogen) and 160 µl phenol/chloroform/isoamyl alcohol (25 : 24 : 1), according to the manufacturer's instructions. To facilitate RNA precipitation, 200 µg glycogen was added to the sample. The extracted RNA was precipitated from the aqueous phase by adding an equal volume of 2-propanol and washed once with 70 % ethanol. RNA was resuspended in sterile distilled water.
Cloning and sequencing of the VDV-1 genome.
Initially, cDNA was synthesized by using the immunocapture RT-PCR method with some modification to the protocol described by Kokko et al. (1996) and combined with 3' rapid amplification of cDNA ends (RACE) methodology. To this aim, Eppendorf tubes were coated with 100 µl antibody solution containing 1 µg IgG in PBS, pH 7·4, for 2 h at 37 °C, washed three times with PBS and incubated overnight at 4 °C with a few ground mites in PBS in order to trap the virus. The unbound material was removed and the tubes were washed three times with PBS, finally removing all excess buffer by pipetting after a brief centrifugation to collect the liquid at the bottom of the tube. First-strand cDNA was synthesized directly in the tube containing the trapped virus by using SuperScript reverse transcriptase (RT) (Invitrogen) and an oligo-dT anchor primer (5'-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV-3'; Roche), assuming that if the isolated virus was indeed an insect picorna-like virus, a poly(A) tail would be present. The first-strand cDNA obtained was used in a PCR using a degenerate forward primer (5'-ATIGTIIIITAYGGIGAYGA-3') for a conserved region around the GDD motif (IVXYGDD), which is highly conserved in the RdRp amino acid sequences of picorna(-like) viruses (see Fig. 3a
, region VI) in combination with the oligo-dT primer. The PCR was run for 35 cycles with an annealing temperature of 40 °C for 3 min and elongation at 72 °C for 3 min, with a final elongation of 7 min at 72 °C. In order to walk towards the 5' end of the viral genome, 5' RACE was used repeatedly by using a 5'RACE kit (Roche) in combination with the Expand Long-template PCR system (Roche), essentially as described previously (van Oers et al., 2003
). This strategy involves the use of specific primers in cDNA synthesis and a nested primer in PCR. PCR products were cloned in pGEM-T Easy (Promega) and verified by automated sequencing. Prior to cloning of the 5'-terminal region of the viral genome, the isolated RNA was treated with proteinase K in order to ensure that the genome-linked protein (VPg) was removed. Total RNA (2 µg) was digested with 100 µg proteinase K ml1 in 50 mM Tris/HCl, pH 7·5, 5 mM CaCl2, 0·2 % SDS at 60 °C for 1 h. The proteinase was subsequently removed by phenol/chloroform/isoamyl alcohol extraction. RNA was precipitated from the aqueous phase with an equal volume of 2-propanol and washed once with 70 % ethanol.
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RESULTS |
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The VDV-1 RNA sequence had 84 % nucleotide identity, without the poly(A) tail, to those of DWV (GenBank accession nos AY292384 and AJ489744) and KV (Fujiyuki et al., 2004). In the 5' NTR, there was a deletion of 11 nt in the VDV-1 sequence (compared with DWV and KV sequences), occurring 2 nt before the AUG translational start codon. Four single-nucleotide and two double-nucleotide deletions, as well as two single-nucleotide insertions, were distributed in this region. Overall, the VDV-1 5' NTR was shorter than the corresponding regions of DWV (GenBank accession no. AY292384) (by 22 nt) and KV (by 39 nt). Table 3
shows the percentage nucleotide identities between VDV-1 and the most closely related viruses, generated from an alignment of the sequences. The VDV-1 RdRp nucleotide sequence had approximately 48 % identity to that of SBV. As the nucleotide sequence of VDV-1 was not present in the online databases (GenBank and EMBL) and was sufficiently different from the sequences of DWV and KV, we propose the name Varroa destructor virus 1 for the virus isolated from the mite V. destructor.
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A phylogenetic tree was constructed for the predicted amino acid sequence of the RdRp domain of VDV-1 and 18 related viruses in GenBank. The RdRp tree (Fig. 4) segregated the viruses into their (assigned or proposed) taxonomic groups (Picornaviridae, Iflavirus and Dicistroviridae). The phylogenetic tree also showed that VDV-1 is related most closely to DWV and KV and clusters together with more distantly related members of the genus Iflavirus. VDV-1, DWV, KV and SBV appeared to be related more closely to each other than to IFV, EoPV and PnPV. The functional domains of the helicase and protease regions of VDV-1, DWV and KV were identical except for variations in the flanking regions and were therefore not suitable for phylogenetic analysis.
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Replication of the virus
During replication of RNA viruses with a single-stranded, positive-sense genome, a full-length, complementary, negative-sense RNA is normally transcribed in the host. This serves as the template for multiplication of the genome for packaging into the capsid to form new virion progeny. A selective RT-PCR was set up to detect the presence of the negative-sense RNA strand in V. destructor mites infected with VDV-1 in an effort to determine whether VDV-1 replicates in this species. The primers used to make cDNA were specific for either the negative or the positive strand (see Table 2). Once specific cDNA was synthesized, a PCR was performed using the same pair of primers for both positive- and negative-strand detection, as PCR is not sense-specific. The controls also used this primer pair and were valid for both positive- and negative-sense detection.
Because VDV-1 and DWV have high nucleotide sequence similarity, primer sets specific for each of the two viruses were designed from regions of the genome that showed divergence, in order to discriminate between these two viruses in a replication assay. The specificity of the PCR primers selected was ascertained by PCRs on plasmid templates bearing a cDNA clone of the VDV-1 or DWV genome region to be amplified. A PCR product of 1129 bp was amplified when VDV-1 primers were used in combination with a VDV-1 template (Fig. 5, lane 4), but not with a DWV-1 template (lane 5), and vice versa for DWV primers (Fig. 5
, lanes 9 and 10). Both VDV-1 (Fig. 5
, lane 3) and DWV (lane 8) replicated in V. destructor mites, as seen by RT-PCR amplification of the negative strand. VDV-1 and DWV positive-strand genomes were also detected (lanes 1 and 6), showing that the mite extract contained both viruses. No product was observed when the RT step was omitted (lanes 2 and 7). The RT-PCR product from the negative-sense RNA template was sequenced and confirmed these results.
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DISCUSSION |
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RdRp is used as a reliable protein for the construction of phylogenetic trees for the classification of RNA viruses, as it tends to be highly conserved among related groups (Zanotto et al., 1996; Culley et al., 2003
). The closest relationship to VDV-1 was found with two bee viruses, DWV and KV. As Varroa mites live in close parasitic proximity to bees, the high homology observed with these two bee viruses was not surprising, as they have probably evolved from a common ancestor.
The icosahedral structure of mammalian picornaviruses is assembled from four capsid proteins. Whilst most of the picorna-like viruses that have been characterized have four capsid proteins, only two proteins have been observed in EoPV (Wang et al., 2004) by using SDS-PAGE and three have been described for RhPV (Moon et al., 1998
). Five proteins were detected on the VDV-1 blot (Fig. 2b
) instead of the usual four. The fifth could be an additional structural protein, an incompletely processed precursor or a contaminating cellular protein from the mite.
The VDV-1 polyprotein had one NPGP motif (aa 216219 from the ORF start), which is a conserved picornavirus motif for cleavage at the 2A/2B site (Ryan & Flint, 1997). PnPV has two NPGP sites, one of which has been demonstrated to be active and to define the break between the first and second coat proteins, whereas the other is assumed to be the C-terminal site of the fourth coat protein relative to the ORF start (Wu et al., 2002
). If we assume that this site is also active in the VDV-1 polyprotein, a protein of approximately 25 kDa would be released. This 25 kDa protein alone would have the lowest identity to homologous proteins of DWV and KV, 83 and 81 %, respectively, and could be a target for specific immunodetection of these viruses, as the antiserum against the structural proteins was probably raised from a mixed population of viruses.
N-terminal sequencing of the structural proteins of IFV and PnPV indicated that the region encoding the coat proteins did not start at the beginning of the ORF. For IFV, the coding region (for VP3) started at aa 149 and for PnPV at aa 320, leading to speculation that these two viruses may encode a leader (L) protein, as with some vertebrate picornaviruses (see Fig. 1). Cardioviruses and aphthoviruses have polyproteins that are somewhat different from those of the other picornaviruses, in that their ORFs encode an additional N-terminal L peptide, which precedes the P1 capsid region (Rueckert, 1996
). The picornavirus L protein is an autocatalytic, papain-like protease that releases itself from the nascent polyprotein after translation of the virus genome (Ryan & Flint, 1997
). It cleaves translation initiation factor eIF-4G, severely restricting or shutting off cap-dependent host protein synthesis during infection, thus giving the virus genome a greater chance of being translated via cap-independent initiation from an upstream IRES in the genome RNA (Dvorak et al., 2001
). Gorbalenya et al. (1991)
identified a cysteine/tryptophan pair and a histidine as the active residues in the aphthovirus L proteinase. The polypeptide sequences preceding the capsid region of IFV and PnPV and the comparable region in VDV-1 do not have the defined critical functional motifs to render them active as proteases of the L type and, unless proven otherwise, there is not sufficient information to indicate that these N-terminal parts function as proteases (see also Ghosh et al., 1999
; Wang et al., 2004
).
In this paper, we have demonstrated that VDV-1 replicates in V. destructor mites and that DWV also replicates in this mite. We did not determine whether the mites also contained KV. Previously, a positive reaction in ELISA with antiserum raised against DWV isolated from bees with deformed wing symptoms was observed in Varroa mites (Bowen-Walker et al., 1999). Although we demonstrated that VDV-1 and DWV replicate in the mite population studied, we have not established their co-existence within a single mite. Despite the fact that DWV and KV are related closely to each other [having RNA identities of 97 % (Fujiyuki et al., 2004
) and polyprotein identities of 98 %], much closer than to VDV-1, they are accompanied by clear pathological differences. DWV causes deformity in the wings of bees, whereas KV manifests as aggressiveness in infected worker bees. KV infection does not result in symptoms of deformation of the wings and its RNA has been detected almost exclusively in the brains of aggressive worker honey bees (Fujiyuki et al., 2004
). The bees at the Wageningen University hives have not, so far, exhibited deformed wings, nor are the workers unduly aggressive. The biological properties of VDV-1 in bees are under study and the pathology of VDV-1, in particular its impact on the health of the mite and bee, needs further investigation.
DWV isolates from Pennsylvania, USA (GenBank accession no. AY292384), Italy (AJ489744) and France (AY224602) that have been completely or partially sequenced show 9899 % nucleotide identity to each other and 9697 % identity to KV. The VDV-1 nucleotide sequence displayed 8384 % identity to those of the DWV isolates and KV. Considering the statistics of the molecular data and the absence of symptoms attributed to DWV and KV infection in the Wageningen bees, we conclude that, despite being related closely to these two viruses, VDV-1 is different. The extent of this difference remains to be determined.
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ACKNOWLEDGEMENTS |
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Received 23 July 2004;
accepted 11 August 2004.