Identification of Kashmir bee virus in France using a new RT-PCR method which distinguishes closely related viruses.

A new RT-PCR protocol has been developed, avoiding potential misdiagnosis of Kashmir bee virus (KBV) linked to the use of KBV primers designed originally. The PCR assay validation was realised taking into account the analytical specificity and the PCR detection limit. KBV was detected in a bee sample collected in France from an apparently healthy apiary in 2012. The specificity of the primers was confirmed by sequencing the PCR product. This French sequence clustered into the KBV genotype by phylogenetic analysis, while previous French sequence isolates collected in 2002 belong to the IAPV genotype. These data represent the first detection of KBV in France.

Development and validation of a real-time two-step RT-qPCR TaqMan(®) assay for quantitation of Sacbrood virus (SBV) and its application to a field survey of symptomatic honey bee colonies.

Sacbrood virus (SBV) is the causal agent of a disease of honey bee larvae, resulting in failure to pupate and causing death. The typical clinical symptom of SBV is an accumulation of SBV-rich fluid in swollen sub-cuticular pouches, forming the characteristic fluid-filled sac that gives its name to the disease. Outbreaks of the disease have been reported in different countries, affecting the development of the brood and causing losses in honey bee colonies. Today, few data are available on the SBV viral load in the case of overt disease in larvae, or for the behavioural changes of SBV-infected adult bees. A two-step real-time RT-PCR assay, based on TaqMan(®) technology using a fluorescent probe (FAM-TAMRA) was therefore developed to quantify Sacbrood virus in larvae, pupae and adult bees from symptomatic apiaries. This assay was first validated according to the recent XP-U47-600 standard issued by the French Standards Institute, where the reliability and the repeatability of the results and the performance of the assay were confirmed. The performance of the qPCR assay was validated over the 6 log range of the standard curve (i.e. from 10(2) to 10(8) copies per well) with a measurement uncertainty evaluated at 0.11log10. The detection and quantitation limits were established respectively at 50 copies and 100 copies of SBV genome, for a template volume of 5µl of cDNA. The RT-qPCR assay was applied during a French SBV outbreak in 2012 where larvae with typical SBV signs were collected, along with individuals without clinical signs. The SBV quantitation revealed that, in symptomatic larvae, the virus load was significantly higher than in samples without clinical signs. Combining quantitation with clinical data, a threshold of SBV viral load related to an overt disease was proposed (10(10) SBV genome copies per individual).

Improving molecular discrimination of Nosema apis and Nosema ceranae.

Nosema apis and Nosema ceranae are the causative agents of nosemosis, a contagious honeybee disease that weakens bee colonies. The species are discriminated through several PCR-based methods including a multiplex PCR recommended by the World Organization for Animal Health (OIE). In this study, the OIE protocol was compared to two other PCR protocols using different PCR kits with the same primer pairs as described in OIE. The results showed that the three PCR protocols have similar sensitivity but only the kit dedicated to multiplex PCR could detect small quantities of one Nosema species when greater quantities of the other were also present. However, singleplex PCR methods are currently the most sensitive methods for discerning each species. These results have important implications for epidemiology and the understanding of the disease.

Experimental infection of the honeybee (Apis mellifera L.) with the chronic bee paralysis virus (CBPV): infectivity of naked CBPV RNAs.

Chronic paralysis is an infectious and contagious disease of the honeybee (Apis mellifera L.) and is caused by the chronic bee paralysis virus (CBPV). This disease leads to death in adult bees and is therefore a serious threat for colony health. CBPV is a positive single-stranded RNA virus and its genome is composed of two RNA segments, RNA 1 and RNA 2, 3674 nt and 2305 nt, respectively. Although CBPV shares some characteristics with viruses classified into families Nodaviridae and Tombusviridae, it has not been assigned to any viral taxa yet. The characterisation of CBPV proteins and their functions are needed to better understand the mechanisms of CBPV infection. However, since honeybee cell lines are not yet available, experimental infection of adult bees is the only method currently available to propagate the virus. With the objective of studying CBPV proteins using the viral genome, we used experimental infection in adult bees to evaluate the infectivity of naked CBPV RNAs by direct inoculation. Our results demonstrated that an injection of naked RNAs, ranging from 10(9) to 10(10) CBPV copies, caused chronic paralysis. Bees inoculated with naked RNA showed chronic paralysis signs 5 days after inoculation. Moreover, injected RNAs replicated and generated viral particles. We therefore provide an in vivo experimental model that will be useful tool for further studies by using a reverse genetics system.

Intra-laboratory validation of chronic bee paralysis virus quantitation using an accredited standardised real-time quantitative RT-PCR method.

Chronic bee paralysis virus (CBPV) is responsible for chronic bee paralysis, an infectious and contagious disease in adult honey bees (Apis mellifera L.). A real-time RT-PCR assay to quantitate the CBPV load is now available. To propose this assay as a reference method, it was characterised further in an intra-laboratory study during which the reliability and the repeatability of results and the performance of the assay were confirmed. The qPCR assay alone and the whole quantitation method (from sample RNA extraction to analysis) were both assessed following the ISO/IEC 17025 standard and the recent XP U47-600 standard issued by the French Standards Institute. The performance of the qPCR assay and of the overall CBPV quantitation method were validated over a 6 log range from 10(2) to 10(8) with a detection limit of 50 and 100 CBPV RNA copies, respectively, and the protocol of the real-time RT-qPCR assay for CBPV quantitation was approved by the French Accreditation Committee.

Chronic bee paralysis: a disease and a virus like no other?

Chronic bee paralysis which was called Paralysis is a rather unusual disease caused by a rather unusual virus. In this review, we explore current knowledge of the disease and its etiological agent. Paralysis is the only common viral disease of adult bees whose symptoms include both behavioural and physiological modifications: trembling and hair loss. The disease often affects the strong colonies of an apiary and thousands of dead individuals are then observed in front of the hives. Two sets of symptoms have traditionally been described in the existing literature, but nowadays we can define a general syndrome. The morphology of the Chronic bee paralysis virus (CBPV) particles and the multipartite organisation of the RNA genome are exceptional, as most honey bee viruses are picorna-like viruses belonging to the Iflavirus and Cripavirus genera with symmetric particles and monopartite positive, single-strand RNA genomes. CBPV is currently classified as an RNA virus but is not included in any family or genus. Although it shares several characteristics with viruses in the Nodaviridae and Tombusviridae families, it differs from previously known viruses according to the various demarcation criteria defined by the International Committee on Taxonomy of Viruses (ICTV). Thus, it should be considered as the type species of a new group of positive-strand RNA viruses. The recent sequencing of the complete CBPV genome has opened the way for phylogenetic studies and development of new molecular tools able to detect variable isolates and to quantify genomic loads. This article considers the results of such recent detection tests but also previous studies including: (i) the distribution of CBPV infection within the bees and the hive, (ii) the way the virus spreads and its persistence in the colony environment, and (iii) geographical and seasonal distribution and impact of CBPV infections.

Phylogenetic analysis of the RNA-dependent RNA polymerase (RdRp) and a predicted structural protein (pSP) of the Chronic bee paralysis virus (CBPV) isolated from various geographic regions.

Chronic bee paralysis virus (CBPV) is responsible for chronic paralysis, an infectious and contagious disease of adult honey bees (Apis mellifera L.). The full-length nucleotide sequences of the two major RNAs of CBPV have previously been characterized. The Orf3 of RNA1 has shown significant similarities to the RNA-dependent RNA polymerase (RdRp) of positive single-stranded RNA viruses, whereas the Orf3 of RNA2 encodes a putative structural protein (pSP). In the present study, honey bees originating from 9 different countries (Austria, Poland, Hungary, Spain, Belgium, Denmark, Switzerland, Uruguay and France) were analysed for the presence of CBPV genome. The complete genomic nucleotide sequence of the RdRp (1947bp) and of the pSP (543bp) from 24 honey bee positive samples was determined and the phylogenetic relationship among isolates was investigated. Four distinct genotypes of CBPV were observed.

First detection of Israeli acute paralysis virus (IAPV) in France, a dicistrovirus affecting honeybees (Apis mellifera).

Bee samples were collected in French apiaries that displayed severe losses and mortality during the winter (from November 2007 to March 2008). They were screened for the presence of Israeli acute paralysis virus (IAPV) by using RT-PCR. Five out of 35 surveyed apiaries, located in two different geographical areas, were found positive. This represents the first reported detection of IAPV in France. The specificity of the PCR products was checked by sequencing. The phylogenetic analysis showed that French isolates of IAPV were closely related to a cluster including American and Australian isolates. Nevertheless, most of American isolates previously reported to be associated to Colony Collapse Disorder (CCD) and an Israeli isolate first isolated in 2004 from dead bees were included in another cluster. Since IAPV was detected in only 14% of the affected apiaries, it was not possible to establish a causal link between IAPV and the severe winter losses that occurred.

In situ hybridization assays for localization of the chronic bee paralysis virus in the honey bee (Apis mellifera) brain.

Chronic bee paralysis virus (CBPV) is a common single-stranded RNA virus which may cause significant losses in honey bee colonies. As this virus seems to exhibit neurotropism, an in situ hybridization based method was developed to localize the genomic and antigenomic CBPV RNAs in infected honey bee brains. Double-stranded cDNA probes as well as genomic and antigenomic-specific single-stranded cDNA probes were prepared, using the polymerase chain reaction in presence of labelled d-UTP with non-radioactive digoxigenin. Both genomic and antigenomic RNAs were detected the brain of honey bee infected naturally or artificially. Hybridization signals were obtained in some somata and neuropile regions of the brain. In particular, high signals were observed at the level of the mushroom bodies and central complex, regions that are known to be engaged in higher neuronal functions and in the optic and antennal lobes that are sensorial neuropiles. Thus, the presence of virus at these levels may explain the nervous symptoms observed in infected bees. The in situ hybridization procedure proved to be a useful tool to localize specifically CBPV and may be helpful for understanding the observed symptoms.

Detection of Chronic bee paralysis virus (CBPV) genome and its replicative RNA form in various hosts and possible ways of spread.

Detection of Chronic bee paralysis virus (CBPV) is reported for the first time in two species of ants (Camponotus vagus and Formica rufa) and in Varroa destructor. A quantitative real-time PCR (qPCR) method was used to detect and quantify CBPV in infected bees, ants and mites. A minus-strand-specific RT-PCR was used to assess viral replication. These results suggest a new way by which the infection may be spread and other sites of viral persistence in the close apiary environment.

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

The complete sequences of the two major RNAs of Chronic bee paralysis virus (CBPV) have been determined. RNA 1 (3674nt long) and RNA 2 (2305nt 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.

Improvement of RT-PCR detection of chronic bee paralysis virus (CBPV) required by the description of genomic variability in French CBPV isolates.

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

Evaluation of a real-time two-step RT-PCR assay for quantitation of Chronic bee paralysis virus (CBPV) genome in experimentally-infected bee tissues and in life stages of a symptomatic colony.

A two-step real-time RT-PCR assay, based on TaqMan technology using a fluorescent probe (FAM-TAMRA) was developed to quantify Chronic bee paralysis virus (CBPV) genome in bee samples. Standard curves obtained from a CBPV control RNA and from a plasmid containing a partial sequence of CBPV showed that this assay provided linear detection over a 7-log range (R(2)>0.99) with a limit of detection of 100 copies, and reliable inter-assay and intra-assay reproducibility. Standardisation including RNA purification and cDNAs synthesis was also validated. The CBPV TaqMan methodology was first evaluated by quantifying the CBPV genomic load in bee samples from an experimental infection obtained by topical application. Up to 1.9 x 10(10) CBPV copies per segment of insect body (head, thorax and abdomen) were revealed whereas a lower CBPV genomic load was detected in dissected organs such as mandibular and hypopharyngeal glands, brain and alimentary canal (up to 7.2 x 10(6) CBPV copies). The CBPV genomic loads in different categories of bees from a hive presenting the trembling symptoms typical of Chronic paralysis were then quantified. Significantly higher CBPV loads were found in guard, symptomatic and dead bees (up to 1.9 x 10(13) CBPV copies) than in forager, drones and house bees (up to 3.4 x 10(6) CBPV copies). The results obtained for symptomatic or dead bees support the correlation between high CBPV genomic load and pathology expression. Moreover, the high CBPV genomic load revealed in guard bees highlights the possible pivotal role played by this category of bees in CBPV infection.

Experimental study on the toxicity of imidacloprid given in syrup to honey bee (Apis mellifera) colonies.

Two groups of eight honey bee colonies were fed with two different concentrations of imidacloprid in saccharose syrup during summer (each colony was given 1 litre of saccharose syrup containing 0.5 microg litre(-1) or 5 microg litre(-1) of imidacloprid on 13 occasions). Their development and survival were followed in parallel with control hives (unfed or fed with saccharose syrup) until the end of the following winter. The parameters followed were: adult bee activity (number of bee entering the hive and pollen carrying activity), adult bee population level, capped brood area, frequency of parasitic and other diseases, mortality, number of frames with brood after wintering and a global score of colonies after wintering. The only parameters linked to feeding with imidacloprid-supplemented saccharose syrup when compared with feeding with non-supplemented syrup were: a statistically non-significant higher activity index of adult bees, a significantly higher frequency of pollen carrying during the feeding period and a larger number of capped brood cells. When imidacloprid was no longer applied, activity and pollen carrying were re-established at a similar level for all groups. Repeated feeding with syrup supplemented with imidacloprid did not provoke any immediate or any delayed mortality before, during or following the next winter, whereas such severe effects are described by several French bee keepers as a consequence of imidacloprid use for seed dressing in neighbouring cultures. In any case, during the whole study, mortality was very low in all groups, with no difference between imidacloprid-fed and control colonies. Further research should now address several hypotheses: the troubles described by bee keepers have causes other than imidacloprid; if such troubles are really due to this insecticide, they may only be observed either when bees consume contaminated pollen, when no other sources of food are available, in the presence of synergic factors (that still need to be identified), with some particular races of bees or when colonies are not strong and healthy.