Complete sequence of a picorna-like virus of the genus Iflavirus replicating in the mite Varroa destructor

Juliette R. Ongus1, Dick Peters1, Jean-Marc Bonmatin2, Eberhard Bengsch2,3, Just M. Vlak1 and Monique M. van Oers1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Aggregations of 27 nm virus-like particles were observed in electron microscopy images of sectioned Varroa destructor mite tissue. The scattered occurrence of individual particles and accumulation of the virions in lattices in the cytoplasm gave an apparent indication that the virus replicates in the mite. Sequence analysis of the RNA of the purified virus revealed a genome organization with high similarity to that of members of the genus Iflavirus. Phylogenetic analysis of the polymerase showed that the virus was related most closely to Deformed wing virus (DWV) and Kakugo virus (KV) of bees. The virus has a genome of 10 112 nt without the poly(A) tail, with an overall RNA genome identity of 84 % to those of DWV and KV and has one large ORF, translated into a 2893 aa polyprotein with an amino acid identity of 95 % to those of DWV and KV. The first 1455 nt of the ORF encoding the lower molecular mass structural proteins shows the greatest diversion from those of DWV and KV, with an RNA identity of 79 %, and translates to a polypeptide of 485 aa with an identity of 90 %. The name proposed for this virus is Varroa destructor virus 1 (VDV-1). To determine whether VDV-1 replicates in mites, a selective RT-PCR was done to detect the presence of the negative-sense RNA strand. The virus isolate and the closely related DWV could be discriminated by two primer sets, each specific to one virus. Both viruses replicated in the population of the mite species studied.

The GenBank/EMBL/DDBJ accession number for the nucleotide sequence determined in this work is AY251269.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Invertebrate picorna-like viruses are small, non-enveloped viruses that form an isometric particle. The positive-sense, single-stranded RNA genome is either mono- or dicistronic (van Regenmortel et al., 2000).

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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Fig. 1. Schematic diagram of the genome organization of Picornaviridae, iflaviruses and Dicistroviridae. The genome-linked protein (VPg) on the 5' end has been shown to be present in picorna-like viruses, such as CrPV and DCV (King & Moore, 1988). Structural proteins (VP1–VP4) are shaded. Some picornaviruses (cardioviruses and aphthoviruses) encode a leader protein (L) at the beginning of the ORF. Viruses belonging to these groups have a poly(A) tail on the 3' end of the genome.

 
The dicistronic viruses have recently been accommodated in the new family Dicistroviridae, containing the single genus Cripavirus (Mayo, 2002) with the type species Cricket paralysis virus (CrPV) (Wilson et al., 2000). Other species within this genus include Acute bee paralysis virus (ABPV) (Govan et al., 2000), Aphid lethal paralysis virus (ALPV) (van Munster et al., 2002), Black queen cell virus (BQCV) (Leat et al., 2000), Drosophila C virus (DCV) (Johnson & Christian, 1998), Rhopalosiphum padi virus (RhPV) (Moon et al., 1998), Taura syndrome virus (TSV) of shrimp (Mari et al., 2002), Triatoma virus (TrV) (Czibener et al., 2000), Himetobi P virus (HiPV) (Nakashima et al., 1999) and Plautia stali intestine virus (PSIV) (Sasaki et al., 1998). Members of this family are characterized by having two non-overlapping ORFs that encode either the structural or non-structural proteins. An intergenic region, which functions as an internal ribosome entry site (IRES), separates the two ORFs. In dicistroviruses, the proteins involved in RNA replication and polyprotein processing are encoded by the 5'-proximal ORF, whereas the 3'-proximal ORF encodes the capsid proteins (Leat et al., 2000).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isolation of virus.
V. destructor mites were collected in June 2001 from heavily infested beehives at Wageningen University, The Netherlands. Mites (1 g) were stirred gently in 2 % ethanol overnight. The mites were collected by centrifugation at 5000 r.p.m. in a Sorvall B21 centrifuge for 5 min and a homogenate was prepared in 1 ml 0·01 M potassium phosphate (K2HPO4/KH2PO4) buffer, pH 7·3. Large debris was removed by centrifugation at 5000 r.p.m. for 10 min at 4 °C. The supernatant was centrifuged through a 30 % sucrose solution at 27 000 r.p.m. for 6 h in a Beckman ultracentrifuge to collect the particles. The pellet containing the viruses was resuspended in 1 ml 0·01 M potassium phosphate buffer and centrifuged at 40 000 r.p.m. for 3 h at 4 °C in a discontinuous 10–40 % sucrose gradient. The fraction containing purified virus particles was removed and resuspended in 1 ml 0·01 M potassium phosphate buffer and stored at 4 °C. A sample was examined under an electron microscope.

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 ml–1 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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Fig. 3. Multiple sequence alignment of VDV-1 with picorna-like viruses for RdRp sequences (a) and helicase (b) and protease (c) domains. Conserved regions corresponding to those recognized by Koonin & Dolja (1993) are indicated by bars above the protein alignment. Black shading indicates 100 % sequence identity and dark-grey shading indicates 80 % identity. Numbers at the beginning of the sequences represent the amino acid position from the start of the ORF. Numbers within sequences represent the number of omitted amino acids. The amino acid sequences were deduced from nucleotide sequences, for which the GenBank accession numbers are given in Table 1.

 
Computer-assisted sequence analysis.
The SeqMan software in the program DNASTAR was used to build contiguous data of overlapping clones from which a consensus nucleotide sequence of the virus was derived by using information from at least six clones for every nucleotide. The consensus nucleotide sequence was compared with related sequences in GenBank by using the BLAST tool on the national centre for biotechnology information (ncbi) site and the FASTA tool for similarity searches on the European Molecular Biology Laboratory (EMBL) site. Phylogenetic trees were constructed with the predicted RdRp amino acid sequences from data available for related viruses in GenBank. CLUSTAL_X software was used to create an alignment of the related sequences and the GeneDoc program was used to edit the alignment. Phylogenetic trees were plotted by using the neighbour-joining method. Confidence levels as percentages were estimated by 1000 replicates in a bootstrap analysis using CLUSTAL_X (Thompson et al., 1997). The virus sequences used in this paper and their GenBank accession numbers are listed in Table 1.


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Table 1. Nucleotide sequence GenBank accession numbers and general classification of the picorna(-like) viruses discussed in this paper

 
RT-PCR.
Primers used in the RT-PCR analysis are listed in Table 2. Total RNA was extracted from live mites and 2 µg was used to make cDNA from the negative-sense RNA by using the forward primer VDV-1 FRT. The incubation temperature of the reaction mixture was raised to 55 °C for 2 min prior to the addition of Avian myeloblastosis virus (AMV) RT (Roche) and the mixture was further incubated at the same temperature for 60 min after adding the enzyme. The transcriptase was inactivated at 70 °C for 10 min. AMV RT and the primer were not added to the negative-control mix during reverse transcription. The cDNA was purified with a High Pure PCR Product purification kit (Roche) and PCR amplification was performed with Taq DNA Polymerase (Promega), using the forward primer VDV-1 FRTPCR and the reverse primer VDV-1 RRTPCR. Following a 5 min denaturing step at 94 °C, PCR was carried out for 30 cycles with denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and 2 min elongation at 72 °C. The final elongation step was done for 7 min at 72 °C. The positive PCR control was a plasmid clone of the region of the virus to be amplified. The same plasmid was used as a template in a PCR with corresponding DWV primers DWV FRTPCR and DWV RRTPCR (based on the sequence of GenBank accession no. AY292384) to establish the specificity of the PCR primers used.


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Table 2. Primers used for selective RT-PCR of VDV-1 and DWV

 
In order to show that VDV-1 was present in the mites from which RNA was extracted, cDNA of the positive-sense RNA strand (viral genome) was synthesized by using the reverse primer VDV-1 R and PCR amplification was carried out using the same PCR primers as for detection of the negative-sense strand, VDV-1 FRTPCR and VDV-1 RRTPCR. The DWV reverse primer DWV-R was used to detect the DWV genome and the forward primer DWV FRT to detect the replication of DWV in mites, following the same procedure as for VDV-1. The PCR products of the replication analysis of both viruses in the mites were sequenced with the respective reverse primers VDV-1 RRTPCR and DWV RRTPCR.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus morphology and structural protein profile
The virus particles purified from V. destructor mites were observed by using an electron microscope. They had an isometric appearance and an approximate diameter of 27 nm (Fig. 2a). Western blot analysis of the virus sample using antibodies raised against the purified virus particles revealed five proteins. The antibodies reacted strongly with a protein of approximately 46 kDa (Fig. 2b). Three reactive proteins were in the 17–25 kDa range and one protein had an estimated size of 32 kDa.



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Fig. 2. Virus particles in V. destructor tissue and Western blot analysis. (a) Electron micrograph of picorna-like virus particles in V. destructor histological sections, showing aggregates of isolated virus particles. Bar, 108 nm. (b) Western blot analysis of virus particles isolated from V. destructor using antiserum raised against purified virus. Protein size markers are indicated (kDa). The 46 (VP1) and 32 (VP2) kDa proteins (arrows) were N-terminally sequenced.

 
Genome sequence
Cloning and sequencing of the VDV-1 genome yielded a continuous sequence of 10 112 nt, not including the poly(A) tail. The nucleotide base composition of the genome was 29·21 % A, 32·20 % U, 22·61 % G and 15·98 % C. The use of an oligo-dT reverse primer in RT-PCR, in combination with a forward degenerate primer annealing to a putative conserved YGDD motif in the polymerase region (Fig. 3a), amplified a product of approximately 800 nt, indicating that a poly(A) tail was present at the 3' end of the genome. The sequence of this PCR product revealed part of the coding sequence for an RdRp, homologous to RdRp sequences of picorna-like viruses. The VDV-1 genome had one large ORF (nt 1118–9799) that was translated into a polyprotein. The structural proteins were encoded by the 5' part of the coding sequence, whereas the 3' part encoded the non-structural proteins. The 5' non-translated region (NTR) was 1117 nt and the 3' NTR had a length of 313 nt.

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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Table 3. Percentage nucleotide and amino acid sequence identity and similarity in an alignment between corresponding regions of VDV-1 and those of other iflaviruses

 
Predicted amino acid sequences
Based on the RNA sequence, the VDV-1 non-structural proteins had conserved functional motifs that were characteristic of RdRps, proteases and helicases of viruses in the picorna-like superfamily (Koonin & Dolja, 1993). Eight conserved domains have been identified in RdRp amino acid sequences (Koonin & Dolja, 1993) and these were also present in VDV-1 (Fig. 3a). The three conserved domains in helicase sequences of picorna-like viruses were present in the VDV-1 helicase (Fig. 3b). The protease of VDV-1 has GXCG and GXHXXG motifs, which are also conserved in the 3C-like proteases of other picorna(-like) viruses that were included in the alignment (Fig. 3c). These conserved regions in the protease are consistent with the putative catalytic residues and substrate-binding sites that were reported by Koonin & Dolja (1993). The conservation of the entire VDV-1 polyprotein and the various segments were compared in an alignment of comparable regions of the most closely related viruses and the results are summarized in Table 3.

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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Fig. 4. Phylogenetic analysis of the RdRp domains. Trees were constructed from the alignment of 19 RdRp sequences by using the neighbour-joining method, as reported by Leat et al. (2000). Numbers at nodes represent bootstrap values as percentages estimated by 1000 replicates in an analysis using CLUSTAL_X software. Branch lengths are proportional to relatedness. VDV-1 (underlined) is located in the Iflavirus cluster. Encephalomyocarditis virus (EMCV) and poliovirus were used as an outgroup.

 
Western blot analysis yielded five structural viral proteins (see Fig. 2b). The two large structural proteins, with molecular masses of approximately 46 and 32 kDa, were N-terminally sequenced. Analysis of the 46 kDa protein gave an amino acid sequence of XNPSYQQS (aa 486–493 in the polyprotein) and the sequence XEESXNTTVLDXTTXLQS (aa 902–919) was obtained for the 32 kDa protein. These positions are in agreement with the arrangement of the structural proteins in the IFV genome (Isawa et al., 1998), the type species of the genus Iflavirus. VP1 is positioned N-terminally from VP2, which is located upstream of the helicase (VP1 being the largest capsid protein and VP4 the smallest) (Fig. 1).

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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Fig. 5. RT-PCR with selective primers for VDV-1 (left panel) and DWV (right panel). Amplified fragments had a length of 1129 bp in both cases. In the RT-PCR for VDV-1, PCRs were performed to amplify the positive-sense viral RNA genome (lane 1), as well as the negative-sense RNA (lane 3). A negative control without the RT step (lane 2) and a positive PCR control using a plasmid clone as template (lane 4) were also performed. Specificity of the primers was checked by PCR using a DWV plasmid clone with VDV-1 primers (lane 5). Replication analysis for DWV was carried out in a similar way (right panel) and lanes 6–10 were loaded in the same order as lanes 1–5.

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
VDV-1 has morphological characteristics similar to those of picorna-like viruses and has a positive-sense RNA genome with an organization matching that of members of the genus Iflavirus. The polyprotein translated from the single large ORF (2893 aa) is the same size as those of DWV and KV. Pairwise alignment of the entire VDV-1 and DWV/KV polyproteins resulted in parallel conservation from the N- to the C-terminal end, and was also observed in a similar alignment of the 2987 aa EoPV (Wang et al., 2004) and 2986 aa PnPV (Wu et al., 2002) polyprotein sequences.

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 216–219 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 98–99 % nucleotide identity to each other and 96–97 % identity to KV. The VDV-1 nucleotide sequence displayed 83–84 % 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.


   ACKNOWLEDGEMENTS
 
We thank Zhang Quiansong and Jan van Lent for their help in electron microscopy. We also thank Johan Calis and Willem-Jan Boot for providing the mites and Angela Vermeesch for initial Western blot analysis. Our special thanks go to Joop Groenewegen (deceased 2001) for the initial electron microscopy. This research was performed in co-operation with CNRS, Orléans, France (contract 081.0870.00) and was partially supported by the 1221/97 EC programme.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 23 July 2004; accepted 11 August 2004.