1 Department of Entomology, Penn State University, University Park, PA 16803, USA
2 National Microbiology Laboratory, Health Canada, Winnipeg, Manitoba, Canada R3E 3R2
3 Department of Biochemistry, Penn State University, University Park, PA 16803, USA
4 Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4R2
Correspondence
J. R. de Miranda
joachimdemiranda{at}yahoo.com
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ABSTRACT |
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The GenBank accession numbers for the nucleotide sequences reported in this paper are AY452696 and AY275710.
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INTRODUCTION |
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KBV is serologically and biologically closely related to acute bee paralysis virus (ABPV; Allen & Ball, 1995; Anderson, 1991
). Like KBV it was discovered as a contaminant, during transmission studies of chronic bee paralysis virus (Bailey et al., 1963
) and is extremely lethal to adults and larvae, both by injection and in larger doses by feeding (Bailey et al., 1963
; Nordstrom, 2000
). It is common in seemingly normal, healthy colonies and has been heavily implicated in varroa-induced colony losses, primarily in Europe in the 1980s (Ball, 1985
; Allen et al., 1986
; Ball & Allen, 1988
; Bailey & Ball, 1991
). Varroa can transfer ABPV among adults and pupae with 5080 % efficiency, depending on the sensitivity of the detection method used (Wiegers, 1988
; Ball, 1989
). This efficiency drops with successive transfers and there is no noticeable latent period between acquisition and transmission, which suggests that there is no virus replication in the mite (Wiegers, 1988
). The experimental host range for ABPV includes Apis and Bombus species, but not several non-hymenopteran insects (Bailey & Gibbs, 1964
).
Although KBV and ABPV are closely related they are not identical. ABPV is often associated with paralysis in adult bees whereas KBV generally is not, the VP4 proteins of the ABPV and KBV particles are serologically distinct (Stoltz et al., 1995), and the viruses can be readily distinguished by RT-PCR (Stoltz et al., 1995
; Evans, 2001
). KBV is also more variable than ABPV, as determined by capsid protein profiles and serology (Bailey et al., 1979
; Allen & Ball, 1995
). This may be an innate property of the virus or the result of an adaptation process if A. mellifera was only recently acquired as a new host of KBV (Bailey & Ball, 1991
). Both viruses can coexist within the same colony (Hung et al., 1996
) and even within the same bee (Evans, 2001
), although the implication of this for their classification is unclear. The ABPV genomic sequence has been determined (Govan et al., 2000
), as well as partial sequences of a large number of European and American ABPV isolates (Bakonyi et al., 2002
). Here, we present the complete genome sequence of KBV and compare this to that of ABPV.
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METHODS |
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Cloning and sequencing.
Intact viral genomic RNA was isolated from the particles and used to prepare cDNAs, employing both random and sequence-specific primers. cDNA fragments were cloned either directly or after PCR amplification with specific primers into one of several plasmid cloning vectors (pBlueScript-II, Stratagene; pGEM-T Easy, Promega; pCR2.1, Invitrogen) and were subsequently sequenced. The 3' terminus of the KBV genome was obtained by cloning oligo(dT) primed cDNA (cDNA kit; Clontech), since the virus group to which KBV belongs naturally has a polyadenylated tail at the 3' end. The 5' end of the KBV genome was obtained by three independent rounds of nested 5' RACE, using the SMART-Oligo protocol (Clontech) and three different cDNA primers. A total of 15 5' RACE clones from these reactions were sequenced.
Protein expression.
Ten fragments of the KBV genome, covering the two major open reading frames (ORF), were cloned into bacterial protein expression plasmids (pQE30-32; Qiagen) behind an N-terminal 6xHistidine tag. The polypeptides thus produced were purified by their histidine tags on Ni-NTA columns. Production and purification of the expressed proteins was confirmed by ELISA, using a horseradish peroxidase (HRP) conjugated anti-(6xHis) monoclonal antibody (A7058; Sigma).
ELISA.
Direct ELISA was performed using 15 ng purified virus or expressed virus protein, 1 : 1000 dilution of primary antibody, 40 ng HRP conjugated protein-A ml1 (P8651; Sigma) as secondary antibody and 33'55' tetramethyl benzidine as the colorimetric substrate (Harlow & Lane, 1988). The reaction was terminated with 3 M H2SO4 and read at 405 nm.
Antisera.
Bee virus antisera used in the ELISA assays were obtained from various sources. The KBV-VP4 and ABPV-VP4 antisera were produced in rabbits using the SDS-PAGE purified VP4 proteins of KBV and ABPV as antigens and are described in Stoltz et al. (1995). The DWV-VP1, BQCV-VP3 and SPV-VP1/2 antisera were similarly produced using SDS-PAGE purified VP1 of deformed wing virus (DWV), VP3 of black queen cell virus (BQCV) and VP1 and VP2 of slow paralysis virus (SPV) as antigens. The DWV-VP1 antiserum is described by de Miranda et al. (2002)
. The KBV-virus, BQCV-virus, CBPV-virus and SBV-virus antisera are a gift of D. Anderson (CSIRO, Canberra, Australia) and were produced in rabbits against purified virus preparations of KBV, BQCV, chronic bee paralysis virus (CBPV) and sacbrood virus (SBV), respectively. The ABPV-IgG antiserum was a gift of I. Fries (Swedish Agricultural University, Uppsala, Sweden) and comprises the IgG fraction of an antiserum prepared in rabbits against purified ABPV.
Sequence analysis.
Each nucleotide position of the KBV sequence reported here is a consensus of at least three independent clones. The consensus sequences of the Canadian and Pennsylvania isolates of KBV have been deposited at GenBank under accession numbers AY452696 (KBV-ca) and AY275710 (KBV-pa).
Phylogenetic analysis and taxonomy.
Two multiple sequence alignments, of 44 and 45 homologous, partial ABPV-KBV sequences, were used for phylogenetic analyses. The first alignment is located in the RNA polymerase region and covers nt ABPV(53065719)/KBV(54065819), while the second alignment, located in the coat protein region, covers nt ABPV(81218519)/KBV(81648562). The alignments were assembled using CLUSTAL W (Thompson et al., 1994) and the cricket paralysis virus (CrPV) sequence (Wilson et al., 2000
) as an outgroup. Phylogenetic inference and bootstrapping followed maximum-parsimony criteria and heuristic search methods as implemented by PAUP (4.0b10; Swofford, 1998
). Poorly supported branches were collapsed.
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RESULTS |
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Analysis of the sequence reveals that KBV is a cricket paralysis-like virus (family Dicistroviridae: genus Cripavirus), whose genome consists of a single positive-strand RNA containing two ORFs, separated by an intergenic region and flanked by untranslated regions. KBV has roughly 70 % nt and aa identity to ABPV across the genome. The 5' non-translated region (5' NTR; nt 1608) is the part of the genome that is least conserved between ABPV and KBV, with only 42 % nt identity. Although the KBV and ABPV 5' NTR are of similar length, there are many gaps in the alignment, and the KBV sequence extends about 116 nt beyond the 5' terminus of ABPV (Fig. 2a). The alignment of this region is anchored by a short, highly conserved stretch of 44 nt (shaded in Fig. 2a
). The intergenic region (Fig. 2b
; nt 64116764) and the 3' NTR (Fig. 2c
; nt 92979506) are both highly conserved between ABPV and KBV at 79 and 76 % nt identity, respectively. The KBV intergenic region is considerably longer than that of ABPV (shaded area in Fig. 2b
). However, it may be shorter than indicated, depending on whether the KBV structural polyprotein is initiated at the first available AUG of the ORF (as shown in Fig. 2b
) or at a prior non-AUG initiation site, as is the case for many other cripaviruses (Johnson & Christian, 1998
; Sasaki & Nakashima, 2000
; Wilson et al., 2000
; Czibener et al., 2000
; van Munster et al., 2002
; Nishiyama et al., 2003
; Domier & McCoppin, 2003
). The larger ORF is located in the 5' half of the genome and contains the three helicase domains, the 3C-protease domain and eight RNA polymerase domains (underlined in Fig. 2d
) identified by Koonin & Dolja (1993)
. The helicase domains include the putative NTP-binding residues 560GxxGxGKS567 and 617DD618 in domains A and B, respectively (Gorbalenya & Koonin, 1989
). The 3C-protease domain conserves the cysteine protease motif 1335GxCG1338 and the putative substrate-binding residues 1354GxHxxG1359. C1337 is the third residue of the protease catalytic triad that also involves a histidine residue and either an aspartate or glutamate residue (Koonin & Dolja, 1993
), possibly H1203 and E1245, by analogy with ABPV (Govan et al., 2000
). The RNA polymerase region contains the universal polymerase motif, 1783YGDD1786, in the pol-VI domain. The shorter ORF is located towards the 3' end of the genome and has two picornavirus capsid protein domains (underlined in Fig. 2e
).
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There were 93 unique nucleotide variations among the KBV-pa sequence clones, appearing uniformly across the genome at a frequency of 1·7x103 per nucleotide sequenced. The vast majority (75 %) of these changes were transitions (CU and AG changes). High transition frequencies are common for RNA viruses and can be explained in part by the relative stability of the G-U pairing in RNA molecules during replication. A high percentage (50 %) of the nucleotide variations within the coding regions result in amino acid changes.
The results of the phylogenetic analyses of the relationships among a range of KBV and ABPV isolates are shown in Fig. 3, for both a section of the polymerase region and the coat protein region. These analyses are similar to those of Evans (2001)
and Bakonyi et al. (2002)
, but with the inclusion of more isolates. The isolates are classified by their original designation in GenBank (KBV or ABPV) and by their geographical origin, with the total number of isolates falling within each geographical group indicated in parentheses. The main observation is that the ABPV and KBV designated isolates are cleanly separated into their respective clades, with the exception of two solitary isolates from Hungary (one for the polymerase region and one for the coat protein region) that fall outside the centres' of the ABPV and KBV clades, as defined by the majority of the isolates. In the polymerase dataset, this isolate was classified as KBV (GenBank accession no. AF468967) while in the coat protein dataset the wayward isolate was classified as ABPV (AF346301).
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DISCUSSION |
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The comparison of closely related viruses such as KBV and ABPV is particularly useful for identifying regions of greater or lesser variability within the genome. For example, although there is great overall conservation within the RNA polymerase region there is a short section between pol-V and pol-VI where variation is either allowed to exist or perhaps even selected for (Fig. 2d). The helicase domains, embedded in a very conserved stretch of sequence, are followed by a highly variable protein sequence and there is also a highly variable region following the second capsid protein domain of the structural polyprotein. The biological functions of these variable proteins is of great interest since they may indicate unique molecular and biological properties of the viruses.
The phylogenetic analyses revealed that KBV and ABPV are distinct viruses and are found in several continents (Allen & Ball, 1996). Both KBV and ABPV isolates have only a limited geographical identity. Isolates of each virus can be broadly separated by their continent of origin, but it is more difficult to identify regional trends within each continent. The most obvious explanation for this is that the KBV and ABPV isolates lose their regional identity through the long distance transport of live bees (packages and migratory beekeeping) within each continent. An alternative explanation is that these virus isolates are united by biological rather than geographical criteria, the most obvious of which is the genetic background of the bees. Despite the considerable international trade in queen bees, regional preferences for certain strains of bees persist, which could account for the continental distinctions between the virus isolates. Both geography and host genetics were used to explain the relationships between sacbrood virus isolates collected from around the world, and from both A. mellifera and A. cerana (Grabensteiner et al., 2001
).
What remains to be determined is whether ABPV, KBV and other variants within this group are autonomous viruses meriting species status or strains of single species (van Regenmortel et al., 2000). Since there is no clear geographical, temporal or ecological separation between KBV and ABPV, species designation will depend heavily on the unique biological and molecular characteristics of these viruses, such as the specificity of virus replication and encapsidation, to support their clear phylogenetic separation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Allen, M. F. & Ball, B. V. (1996). The incidence and world distribution of honey bee viruses. Bee World 77, 141162.
Allen, M. F., Ball, B. V., White, R. F. & Antoniw, J. F. (1986). The detection of acute paralysis virus in Varroa jacobsoni by the use of a simple indirect ELISA. J Apic Res 25, 100105.
Anderson, D. L. (1991). Kashmir bee virus a relatively harmless virus of honey bee colonies. Am Bee J 131, 767770.
Anderson, D. L. & Gibbs, A. J. (1988). Inapparent virus infections and their interactions in pupae of the honey bee (Apis mellifera Linnaeus) in Australia. J Gen Virol 69, 16171625.
Bailey, L. & Ball, B. V. (1991). Honey Bee Pathology, 2nd edn. London: Academic Press.
Bailey, L. & Gibbs, A. J. (1964). Acute infection of bees with paralysis virus. J Insect Pathol 6, 395407.
Bailey, L. & Woods, R. D. (1977). Two more small RNA viruses from honey bees and further observations on sacbrood and acute bee-paralysis viruses. J Gen Virol 37, 175182.
Bailey, L., Gibbs, A. J. & Woods, R. D. (1963). Two viruses from adult honey bees (Apis mellifera Linnaeus). Virology 21, 390395.[CrossRef]
Bailey, L., Carpenter, J. M. & Woods, R. D. (1979). Egypt bee virus and Australian isolates of Kashmir bee virus. J Gen Virol 43, 641647.
Bakonyi, T., Grabensteiner, E., Kolodziejek, J., Rusvai, M., Topolska, G., Ritter, W. & Nowotny, N. (2002). Phylogenetic analysis of acute bee paralysis virus strains. Appl Environ Microbiol 68, 64466450.
Ball, B. V. (1985). Acute paralysis virus isolates from honeybee colonies infested with Varroa jacobsoni. J Apic Res 24, 115119.
Ball, B. V. (1989). Varroa jacobsoni as a virus vector. In Present Status of Varroatosis in Europe and Progress in the Varroa Mite Control, pp. 241244. Edited by R. Cavalloro. Rotterdam: A. A. Balkema.
Ball, B. V. & Allen, M. F. (1988). The prevalence of pathogens in honey bee (Apis mellifera) colonies infested with the parasitic mite Varroa jacobsoni. Ann Appl Biol 113, 237244.
Ball, B. V. & Bailey, L. (1997). Viruses. In Honey Bee Pests, Predators and Diseases, 3rd edn, pp. 1131. Edited by R. A. Morse & K. Flottum. Medina, OH: A. I. Root.
Czibener, C., La Torre, J. L., Muscio, O. A., Ugalde, R. A. & Scodeller, E. A. (2000). Nucleotide sequence analysis of Triatoma virus shows that it is a member of a novel group of insect RNA viruses. J Gen Virol 81, 11491154.
de Miranda, J. R., Shen, M. & Camazine, S. M. (2002). Molecular characterization of deformed wing virus. In Proceedings of the 2nd International Conference on Africanized Honey Bees and Bee Mites, pp. 265270. Edited by E. H. Erickson, R. E. Page & A. A. Hanna. Medina, OH: A. I. Root.
Domier, L. L. & McCoppin, N. K. (2003). In vivo activity of Rhopalosiphum padi virus internal ribosome entry sites. J Gen Virol 84, 415419.
Evans, J. D. (2001). Genetic evidence for coinfection of honey bees by acute bee paralysis and Kashmir bee viruses. J Invertebr Pathol 78, 189193.[CrossRef][Medline]
Gorbalenya, A. E. & Koonin, E. V. (1989). Viral proteins containing the purine NTP-binding sequence pattern. Nucleic Acids Res 17, 84138440.[Abstract]
Govan, V. A., Leat, N., Allsopp, M. & Davison, S. (2000). Analysis of the complete genome sequence of acute bee paralysis virus shows that it belongs to the novel group of insect-infecting RNA viruses. Virology 277, 457463.[CrossRef][Medline]
Grabensteiner, E., Ritter, W., Carter, M. J. & 8 other authors (2001). Sacbrood virus of the honeybee (Apis mellifera): rapid identification and phylogenetic analysis using reverse transcription-PCR. Clin Diagn Lab Immunol 8, 93104.
Gromeier, M., Wimmer, E. & Gorbalenya, A. E. (1999). Genetics, pathogenesis and evolution of picornaviruses. In Origin and Evolution of Viruses, pp. 287343. Edited by E. Domingo, R. G. Webster & J. J. Holland. London: Academic Press.
Harlow, E. & Lane, D. P. (1988). Antibodies: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Hung, A. C. F., Ball, B. V., Adams, J. R., Shimanuki, H. & Knox, D. A. (1996). A scientific note on the detection of American strains of acute paralysis virus and Kashmir bee virus in dead bees in one US honeybee (Apis mellifera L.) colony. Apidologie 27, 5556.
Johnson, K. N. & Christian, P. D. (1998). The novel genome organization of the insect picorna-like virus Drosophila C virus suggests this virus belongs to a previously undescribed virus family. J Gen Virol 79, 191203.[Abstract]
Koonin, E. V. & Dolja, V. V. (1993). Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Crit Rev Biochem Mol Biol 28, 375430.[Abstract]
Nishiyama, T., Yamamoto, H., Shibuya, N., Hatakeyama, Y., Hachimori, A., Uchiumi, T. & Nakashima, N. (2003). Structural elements in the internal ribosome entry site of Plautia stali intestine virus responsible for binding with ribosomes. Nucleic Acids Res 31, 24342442.
Nordstrom, S. (2000). Virus infections and varroa mite infestations in honey bee colonies. PhD thesis. Swedish University of Agricultural Sciences, Uppsala, Sweden.
Sasaki, J. & Nakashima, N. (2000). Methionine-independent initiation of translation in the capsid protein of an insect RNA virus. Proc Natl Acad Sci U S A 97, 15121515.
Stoltz, D., Shen, X.-R., Boggis, C. & Sisson, G. (1995). Molecular diagnosis of Kashmir bee virus infection. J Apic Res 34, 153160.
Swofford, D. L. (1998). PAUP* Phylogenetic Analysis Using Parsimony (*and other methods). version 4.0 Sunderland, Massachusetts: Sinnauer Associates.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
van Munster, M., Dullemans, A. M., Verbeek, M., van den Heuvel, J. F., Clerivet, A. & van der Wilk, F. (2002). Sequence analysis and genomic organization of Aphid lethal paralysis virus: a new member of the family Dicistroviridae. J Gen Virol 83, 31313138.
van Regenmortel, M. H. V., Fauquet, C. M., Bishop, D. H. L. & 8 other editors (2000). Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses. San Diego: Academic Press.
Wiegers, F. P. (1988). Transmission of honeybee viruses by Varroa jacobsoni Oud. In European Research on Varroatosis Control, pp. 99104. Edited by R. Cavalloro. Rotterdam: A. A. Balkema Publishers.
Wilson, J. E., Powell, M. J., Hoover, S. E. & Sarnow, P. (2000). Naturally occurring dicistronic cricket paralysis virus RNA is regulated by two internal ribosome entry sites. Mol Cell Biol 20, 49904999.
Received 21 January 2004;
accepted 26 April 2004.