Sacbrood Virus of the Honeybee ( Apis mellifera ): Rapid Identification and Phylogenetic Analysis Using Reverse Transcription-PCR

Abstract

Sacbrood virus (SBV) infects larvae of the honeybee (Apis mellifera), resulting in failure to pupate and death. Until now, identification of viruses in honeybee infections has been based on traditional methods such as electron microscopy, immunodiffusion, and enzyme-linked immunosorbent assay. Culture cannot be used because no honeybee cell lines are available. These techniques are low in sensitivity and specificity. However, the complete nucleotide sequence of SBV has recently been determined, and with these data, we now report a reverse transcription-PCR (RT-PCR) test for the direct, rapid, and sensitive detection of these viruses. RT-PCR was used to target five different areas of the SBV genome using infected honeybees and larvae originating from geographically distinct regions. The RT-PCR assay proved to be a rapid, specific, and sensitive diagnostic tool for the direct detection of SBV nucleic acid in samples of infected honeybees and brood regardless of geographic origin. The amplification products were sequenced, and phylogenetic analysis suggested the existence of at least three distinct genotypes of SBV.

Multiple virus infections in the honey bee and genome divergence of honey bee viruses

“…The polymerase chain reaction (PCR) assay has revolutionized the diagnosis of virus infection and offers a standard method for the specific and sensitive diagnosis of virus infection. Several studies used a RT-PCR assay to detect and identify virus infections in honey bee colonies (Bakonyi et al, 2002;Benjeddou et al, 2001;Evans, 2001;Grabenstiner et al, 2001;Hung et al, 2000;Ribiere et al, 2002;Stoltz et al, 1995). Nevertheless, single RT-PCR assay allows the detection of only one virus per reaction and detection of mixed virus infections would require several separate RT-PCRs.…”

Genomic characterization and phylogenetic analysis of Chinese sacbrood virus isolated from Loess Plateau, China

“…CSBV-SX contained a base composition very similar to that of other SBV strains, including CSBV-BJ, CSBV-GZ, SBV-UK, and SBV-19 (Ghosh et al, 1999;Grabensteiner et al, 2001;Zhang et al, 2001;Chen et al, 2006;Mingxiao et al, 2011;Choe et al, 2012). Genomic alignment analysis showed that CSBV-SX shared high homology with other SBV strains (88.3-97.5%), and that it was classified into the same group as the CSBV-BJ strain in the complete genomic phylogenetic analysis.…”

“…Reverse transcription-PCR was successfully used to identify the RNA genomic virus infection, and nested or seminested PCR was used in these identification procedures (Grabensteiner et al, 2001;Choe et al, 2012;Reddy et al, 2013). For nested PCR or seminested PCR, at least 2 rounds of PCR amplification were needed to complete and verify the identification, which were followed by nucleotide sequencing.…”

A severe sacbrood virus outbreak in a honeybee (Apis mellifera L.) colony: a case report

“…Nevertheless, changing the queen could be of benefit to the hygienic behaviour of the colony or could limit the vertical transmission of the disease (Chen et al 2006). Moreover, genetic variations of the 'European genotype' of SBV have been reported suggesting distinct strains of the virus (Grabensteiner et al 2001). However, little information is available concerning the virulence of these different viral strains.…”

“…No data are available regarding the prevalence of the SBV disease in France but the majority of infections probably remain asymptomatic given the very large spread of the virus. In France and elsewhere, some authors have reported overt infection in brood or SBV detection in colonies without clinical symptoms (Grabensteiner et al 2001;Tentcheva et al 2004;Antunez et al 2006;Nielsen et al 2008;Blanchard et al 2014). Larvae infected by this Iflaviridae (King et al 2011) fail to pupate four days after they have been sealed in their brood-comb cells by the worker bees (White 1917;Bailey et al 1964).…”