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    5 Philippe Blanchard*, Violaine Olivier, Anne-Laure Iscache, Olivier 6 Celle, Frank Schurr, Perrine Lallemand, Magali Ribière. 7

    8 Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Les Templiers, Route

    9 des Chappes, BP 111, 06902 Sophia Antipolis, France. 10





    15 *Corresponding author

    16 Mailing address: Blanchard Philippe, AFSSA Sophia-Antipolis, Unité Pathologie de

    17 l’Abeille, Les Templiers, BP 111, Fr – 06902 Sophia Antipolis

    18 Telephone number: +33 (0) 492.943.726

    19 Fax number: +33 (0) 492.943.701

    20 e-mail:


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22 Abstract

    23 A new RT-PCR test has been developed to diagnose Chronic bee paralysis virus (CBPV), 24 able to detect genetically variable viral isolates. In fact, up to 8.7% divergence between 25 partial nucleotide sequences from viral isolates from French honey bees was highlighted 26 in a preliminary variability study. The previously-described RT-PCR was unable to detect 27 all these viral isolates and RT-PCR diagnosis needed improvement. The new RT-PCR 28 test allow to detect up to 40% of extra CBPV isolates.


    30 Keywords: Chronic bee paralysis virus (CBPV), RT-PCR diagnosis, Genomic variability, 31 Apis mellifera.


    33 Short communication

    34 Chronic paralysis is known to induce significant losses in honey bee colonies (Allen & 35 Ball, 1996;Ball & Bailey, 1997). The pathology is characterized by clusters of trembling, 36 flightless, crawling bees and by individual bees, sometimes hairless, standing at the hive 37 entrance (Bailey et al., 1983). Current diagnosis of the clinical disease is based on an 38 Agarose Gel ImmunoDiffusion (AGID) test (Ball, 1999; Ribière et al., 2000),

    39 complemented by RT-PCR (Ribière et al., 2002). First isolated in 1963 (Bailey et al.,

    40 1963), the aetiological agent of this pathology is the Chronic bee paralysis virus (CBPV), 41 which consists of a single stranded RNA genome. Only a short fragment of the viral 42 genome sequence has been reported to date (Ribière et al., 2002) and research to

    43 sequence the complete CBPV genome is currently in progress in our laboratory. 44 Samples of trembling and crawling bees from apiaries in different French departments 45 were recently subjected to AGID and RT-PCR tests. Although the AGID test gave positive 46 results, no amplification was obtained with RT-PCR. The genomic variability between 47 partial sequences of CBPV from several bee samples was indicated. Genomic variability 48 in RNA viruses has been observed widely (Domingo et al., 1996). This study led us to

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    49 design new RT-PCR primers able to detect all these viral samples and thereby improve 50 the CBPV diagnosis.

    51 In this paper, we describe the development of a new RT-PCR diagnostic test for the 52 detection of CBPV isolates presenting genomic variability. The 5’ RACE System 53 (Invitrogen) was used on the reference isolate (Isolate A) to obtain an additional 54 sequence from the one reported previously (Ribière et al., 2002). Then, a preliminary

    55 study of the genomic variability of seven CBPV viral isolates was begun. Bee samples 56 were collected from honey bee colonies presenting typical symptoms of chronic paralysis, 57 located in the French departments of Aisne, Alpes-Maritimes, Drôme, Essonne and 58 Rhône. Sample preparation, RNA extraction and cDNA synthesis were performed as 59 described previously (Ribière et al., 2002; Blanchard et al., 2007). Several primers were

    60 tested and two sets of primers were retained, allowing to obtain by assembling a 61 sequence of 1285 bases for each isolate. Using these 2 sets of primers, a next PCR was

    ;62 performed with a high-fidelity DNA polymerase (Platinum Pfx DNA Polymerase,

    63 Invitrogen). The PCR products were purified using the Qiaquick PCR purification kit 64 (Qiagen) and sequenced (Millegen, France). The 1285 bp sequence, located in the 65 putative viral RNA-dependent RNA polymerase (RdRp) gene region of CBPV, was 66 aligned using the Clustal W method in MegAlign in the Dnastar package. 67 Isolate A is the CBPV reference isolate obtained by infectivity test as previously 68 described (Ribière et al., 2000). Isolate B is the sequence obtained from field samples

    which presented the most variability with the reference isolate. Analysis of sequence pair 69

    70 distances using the Clustal W method revealed 8.7% divergence between the two 71 sequences, mainly at the 3’ region of this sequence whereas analysis of the amino acid 72 sequence revealed only 2.5% divergence between the two sequences. The alignment of 73 the sequence of the two isolates is illustrated in Figure 1.

    74 These initial observations demonstrated the nucleotide sequence variability of the 3’ 75 region, where primer pair CBPV 1 and CBPV 2 used for the first RT-PCR test was 76 designed (Ribière et al., 2002), revealing 3 and 4 mismatches respectively in each primer.

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    77 A new RT-PCR test 2 using the primer pair designated CBPV 1-1 and CBPV 1-2 was 78 designed by Primer3 Output and targeted the most highly conserved nucleotide region. 79 The sequences, orientations and locations of these two oligonucleotide primer sets for 80 the detection of CBPV by RT-PCR and the sizes of each amplification product are 81 presented in Table 1 and in Figure 1. Amplifications were carried out in 25 µl total

    ;82 reaction volume using the Platinum Taq DNA Polymerase (Invitrogen). The final PCR

    83 reaction contained 20 mM of Tris-HCl pH 8.4, 50 mM of KCl, 1.5 mM of MgCl2, 0.2 mM of

    ;84 dNTPs mix PCR grade, 0.4 µM of each primer, 2.5 units of Platinum Taq DNA

    85 Polymerase and 2.5 µl of cDNA synthesised as described by Ribière et al. (2002). The

    86 thermal cycling conditions were 1 min at 95?C (denaturing of the template and activation 87 of the enzyme), followed by 30 cycles consisting of denaturing at 95?C for 30 sec, 88 annealing at 55?C for 30 sec and extension at 72?C for 1 min, completed by a final 89 elongation at 72?C for 5 min. Negative controls (RT and PCR controls) were included. 90 The PCR products were then electrophoresed in 1% agarose gel in TAE buffer, stained 91 with ethidium bromide, and visualised under UV light (Figure 2). No amplification was 92 detected in the negative controls of either RT-PCR test (N and Nfor RT control and N’ 12 1

    93 and N’for PCR control). Only the CBPV reference isolate and two of the seven tested 2

    94 isolates were detected with RT-PCR test 1 using primers CBPV 1 and CBPV 2, whereas 95 all the isolates including isolate B (lane B) could be detected with RT-PCR test 2 using 2

    96 primers CBPV 1-1 and CBPV 1-2. These results confirm the performance of the RT-PCR 97 test 2, which is able to detect viral isolates, even if they present genomic variability. The

    498 limit of detection of CBPV by this new RT-PCR test was of 10 CBPV copies per reaction

    99 which is identical to the one of the RT-PCR test 1, however lower than the limit of 100 detection of the CBPV TaqMan PCR previously determined at 100 copies (Blanchard et 101 al, 2007).

    102 The genomic variability highlighted in this preliminary study of several French CBPV 103 isolates showed 8.7% divergence between partial sequences obtained from these 104 isolates and located in the putative viral RNA polymerase gene region. However, this

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    105 nucleotide sequence variability did not strongly affect the amino acid sequence of the 106 transcribed RdRp protein. Sequencing of the complete CBPV genome sequence, 107 consisting of two main positive sense single stranded RNAs (RNA 1 and RNA 2) (Overton

    108 et al., 1982), is currently in progress in our laboratory. Preliminary results for genomic 109 variability of these two RNAs confirmed the variability between isolates A and B, and 110 revealed respectively 9.2 and 8.7% of divergence. CBPV detection by RT-PCR had to be 111 improved to take this genomic variability into account. The RT-PCR test 2, described in 112 this paper, is able to detect different viral isolates. To confirm these results, a 113 complementary study was carried out to determine the proportion of isolates not detected 114 by the RT-PCR test 1, allowing to assess the percentage of CBPV genomic variability. 115 This study concerned 70 samples coming from France and European countries. All 116 samples were detected CBPV positive with the second test (CBPV 1-1 / CBPV 1-2), while 117 only 42 samples were detected CBPV positive with the first test (CBPV 1 / CBPV 2), 118 indicating that nearly 40% of the samples are genetically divergent of the reference 119 isolate. At the same time, a real-time two-step RT-PCR assay was developed, using a 120 primers and probe set located in Figure 1 and able to detect and quantify such isolates

    121 (Blanchard et al., 2007).

    122 Genomic variability has already been described on isolates from different countries for 123 honeybee viruses such as Acute bee paralysis virus (Bakonyi et al., 2002) and Sacbrood

    124 virus (Grabensteiner et al., 2001). We are currently carrying on the study of genomic 125 variability of CBPV strains collected from different countries in Europe and worldwide. 126 This study of isolates from several countries should validate the reliability of the RT-PCR 127 test and sequencing of these isolates will permit phylogenetic analyses, assessing the 128 genetic relationships between strains of different geographic origins. 129

    130 Acknowledgements

    131 This work was supported by the French Ministère de l’Agriculture et de l’Alimentation and

    132 with funds from the “Fonds Européens d’Orientation et de Garantie Agricole“ (FEOGA), in

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    133 accordance with the French programme for the improvement of the production and 134 commercialisation of beekeeping products. We are grateful to the collaborators and 135 beekeepers for having kindly provided bee samples.


    137 Captions to figures

    138 Figure 1.

    139 Alignment of the nucleotide sequences (1285b) of the two CBPV isolates: isolate A is the 140 reference isolate and isolate B, the one presenting the most variability. Nucleotides 141 differing between the sequences are indicated in white. The two primer sets used in RT-142 PCR tests (CBPV 1 and CBPV 2 for RT-PCR test 1 and CBPV 1-1 and CBPV 1-2 for RT-143 PCR test 2) and the primers (qCBPV forward, qCBPV reverse) and probe (qCBPV probe)

    144 set used in quantitative real-time PCR described by Blanchard et al. (2007) are located. 145

    146 Table 1.

    147 Primers used for CBPV RT-PCR tests, CBPV 1 and CBPV 2 for RT-PCR test 1 (Ribière

    148 et al., 2002), CBPV 1-1 and CBPV 1-2 for RT-PCR test 2 (this article). 149

    150 Figure 2.

    151 Visualisation of the PCR products obtained with the RT-PCR test 1 (Ribière et al., 2002)

    152 and RT-PCR test 2, amplifying PCR fragments of 455 bp and 570 bp respectively. 153 Lane MW: 100 bp DNA Ladder (Invitrogen).

    154 Lane N: RT negative controls.

    155 Lane A: CBPV reference isolate A.

    156 Lane B to lane H: CBPV isolates from different departments.

    157 Lane N’: PCR negative controls.


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159 References


    161 Allen, M. & Ball, B. V., 1996. The incidence and world distribution of honey bee viruses. 162 Bee World 77, 141-162.

    163 Bailey, L., Ball, B. V. & Perry, J. N., 1983. Honeybee paralysis: its natural spread and its 164 diminished incidence in England and Wales. J. Apic. Res. 22, 191-195. 165 Bailey, L., Gibbs, A. J. & Woods, R. D., 1963. Two viruses from adult honey bees (Apis

    166 mellifera Linnaeus). Virol. 21, 390-395.

    167 Bakonyi, T., Grabensteiner, E., Kolodziejek, J., Rusvai, M., Topolska, G., Ritter, W. & 168 Nowotny, N., 2002. Phylogenetic analysis of acute bee paralysis virus strains. 169 Appl. Environ. Microbiol. 68, 6446-6450.

    170 Ball, B. V., 1999. Paralysis. In Bee disease diagnosis, pp. 81-89. Edited by M. E. Colin, B.

    171 V. Ball & M. Kilani: Options Mediterannéennes.

    172 Ball, B. V. & Bailey, L., 1997. Viruses. In Honey bee pests, predators, & diseases, Third

    173 edition, pp. 11-32. Edited by R. A. Morse & K. Flottum. Medina: A.I. Root 174 Company.

    175 Blanchard, P., Ribière, M., Celle, O., Lallemand, P., Schurr, F., Olivier, V., Iscache, A. L. 176 & Faucon, J. P., 2007. Evaluation of a real-time two step RT-PCR assay for 177 quantitation of Chronic bee paralysis virus (CBPV) genome in experimentally-178 infected bee tissues and in life stages of a symptomatic colony. J. Virol. Methods 179 141, 7-13.

    180 Domingo, E., Escarmis, C., Sevilla, N., Moya, A., Santiago, F., Quer, E. J., Novella, I. S., 181 Holland, J.J., 1996. Basic concepts in RNA virus evolution. The FASEB Journal 182 10, 859-864.

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    183 Grabensteiner, E., Ritter, W., Carter, M. J., Davison, S., Pechhacker, H., Kolodziejek, J., 184 Boecking, O., Derakhshifar, I., Moosbeckhofer, R., Licek, E. & Nowotny, N., 2001. 185 Sacbrood virus of the honeybee (Apis mellifera): rapid identification and

    186 phylogenetic analysis using reverse transcription-PCR. Clin. Diagn. Lab. Immunol. 187 8, 93-104.

    188 Overton, H. A., Buck, K. W., Bailey, L. & Ball, B. V., 1982. Relationships between the 189 RNA components of chronic bee-paralysis virus and those of chronic bee-190 paralysis virus associate. J. Gen. Virol. 63, 171-179.

    191 Ribière, M., Faucon, J. P. & Pépin, M., 2000. Detection of chronic bee paralysis virus 192 infection: application to a field survey. Apidologie 31, 567-577. 193 Ribière, M., Triboulot, C., Mathieu, L., Aurières, C., Faucon, J. P. & Pépin, M., 2002. 194 Molecular diagnosis of chronic bee paralysis virus infection. Apidologie 33, 339-195 351.




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    CBPV 1-1

    qCBPV forward qCBPV probe CBPV 1-2 / qCBPV reverse

    CBPV 1

    CBPV 2 199 200

    201 Figure 1


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    204 Figure 2


     RT- PCR 1 RT- PCR 2 206

    MW N A B C D EF G H N’ MW N A B C D EF G H N’ 11111 1 111122222 2 2222

    570 bp

    455 bp

    207 208





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