Molecular characterisation and phylogenetic analysis of Chronic bee paralysis virus, a honey bee virus

Abstract

The complete sequences of the two major RNAs of Chronic bee paralysis virus (CBPV) have been determined. RNA 1 (3674 nt long) and RNA 2 (2305 nt long) are positive single-stranded RNAs that are capped but not polyadenylated. The 3′ ends of both RNAs are unreactive to polymerisation or ligation even in denaturing conditions, a feature already observed in alphanodavirus RNAs. The three previously described smaller RNAs [Overton, H.A., Buck, K.W., Bailey, L., et al., 1982. Relationships between the RNA components of Chronic bee-paralysis virus and those of chronic bee-paralysis virus associate. J. Gen. Virol. 63, 171–179], were not detected in this study, supporting the hypothesis that they would correspond to the three RNAs of the Chronic bee paralysis satellite virus (CBPSV). RNA 1 and RNA 2 encoded three and four overlapping open reading frames (ORFs), respectively. The amino acid sequences deduced from the ORF 3 on RNA 1 shared the conserved motifs of the RNA-dependent RNA polymerase (RdRp) sequence and presented similarities with members of the Nodaviridae and Tombusviridae families. However, no similarities were found between the other CBPV deduced amino acid sequences and sequences in the NCBI databases, suggesting that CBPV is the prototype of a new family of positive single-stranded RNA viruses.

Introduction

Chronic bee paralysis virus (CBPV) is responsible for chronic paralysis, an infectious and contagious disease of adult honey bees (Apis mellifera L.). This disease presents well-defined symptoms including abnormal trembling of the wings and bodies of diseased bees which are unable to fly but often crawl on the ground (Ball, 1999b). Some individuals become almost hairless, and therefore darker in appearance, and suffer nibbling attacks by the healthy bees in their colony. Diseased bees die within a few days. CBPV can persist as an unapparent infection but may multiply to high levels in honey bees (Blanchard et al., 2007a) and cause significant losses in colonies (Allen and Ball, 1996). Nutritional deficiency, severe winters or bad weather conditions in summer may favour disease outbreaks (Allen and Ball, 1996, Bailey et al., 1983). The distribution of this virus is worldwide (Allen and Ball, 1996), most likely resulting from the intensive commercial exchange of honey bees. In France, the prevalence of CBPV in selected asymptomatic apiaries is 28% (Tentcheva et al., 2004). Current diagnosis of the clinical disease is based on an agarose gel immuno-diffusion test (AGID) (Ribière et al., 2000), complemented by RT-PCR tests which allow the detection of CBPV even in asymptomatic colonies (Blanchard et al., 2007b, Ribière et al., 2002). Recently, a two-step real time RT-PCR assay was developed to quantify the CBPV genomic load in bees and improve understanding of the dynamics of infection both in individual bees and within the colony (Blanchard et al., 2007a).

CBPV seems to have a nervous tropism. Many millions particles of CBPV can be extracted from one bee with chronic paralysis and about half the numbers of particles are in the head, which is only about one-tenth of the total body weight (Ball, 1999b). CBPV particles have been seen in sections of brain tissue, hypopharyngeal and mandibular ganglia and in abdominal and thoracic ganglia (Giauffret et al., 1966a, Giauffret et al., 1970, Lee and Furgala, 1965). They have also been observed in the hind-gut epithelium (Giauffret et al., 1966b, Giauffret et al., 1967) but not in the fat or muscle tissue of diseased bees (Lee and Furgala, 1965).

CBPV was first isolated from diseased honey bees in 1963 (Bailey et al., 1963). Purified preparations contain anisometric, mostly ellipsoidal particles, 30–65 nm in modal length and about 20 nm in width (Bailey et al., 1968). However a large range of particle shapes and sizes has been observed, including rings, figure of eights, branching rods and lengths up to 640 nm (Ball and Bailey, 1991). The virus particles were first reported to possess a single capsid protein of about 23.5 kDa (Bailey, 1976). Western blot analysis further revealed four CBPV-associated polypeptides of about 75, 50, 30 and 20 kDa (Ribière et al., 2000). The CBPV genome was originally described as containing five single-stranded RNA fragments: two major RNAs, RNA 1 and RNA 2 of about 4200 and 2800 nt, respectively, and three minor RNAs, RNA 3a, 3b and 3c, each of about 1100 nt (Overton et al., 1982). A partial CBPV RNA 1 sequence was submitted to the GenBank database (accession number: AF461053) (Ribière et al., 2002).

Bailey et al. (1980), during CBPV investigations, consistently observed large amounts of a minute isometric particle of 17 nm in diameter, serologically unrelated to CBPV but which multiplied only in its presence (Overton et al., 1982). This particle was first called Chronic bee paralysis virus associate (CBPVA) (Bailey, 1976, Bailey et al., 1980). The genome consisted of three single-stranded RNA fragments: A, B and C of about 1100 nt, which were shown, by polyacrylamide gel analysis, to possess indistinguishable size but different secondary structures (Overton et al., 1982). Fingerprint analysis showed that (i) these RNAs had different sequences, (ii) they seemed identical to the minor CBPV RNAs species, 3a, 3b and 3c and (iii) they presented sequence similarities with CBPV RNA 2 (Overton et al., 1982). CBPVA is the first recorded satellite virus in insects. It has recently been renamed Chronic bee paralysis satellite virus (CBPSV) and is classified as the only member of the Chronic bee paralysis virus associated satellite subgroup which is distinct from the Tobacco necrosis satellite virus-like subgroup. (Fauquet et al., 2005).

The morphology of the CBPV particles and the multipartite organisation of the RNA genome are exceptional, as most honey bee viruses are picorna-like viruses in the Iflavirus and Cripavirus genera with symmetric particles and monopartite positive single-stranded RNA genomes (Olivier and Ribière, 2006). CBPV is currently classified as a RNA virus but is not included in any family or genus.

In this study, we report and analyse the complete nucleotide sequences of the two major CBPV RNAs and predict their molecular organisation. According to the phylogenetic analysis and comparison of characteristics, CBPV is distant from any previously defined viral family although the conserved domains of its RdRp depict similarities with those of members of the Nodaviridae and Tombusviridae families. The overall results of this study suggest that CBPV should be considered as the prototype of a new virus family with a segmented positive-stranded RNA genome.