Molecular detection and characterisation of RNA viruses of honeybees

Abstract

Honeybees have evolved through the centuries to inhabit most parts of the world except for the extreme Polar Regions. These insects have also been susceptible to pathogens and disease which has always been part of the honeybees’ ecology and has evolved and adapted accordingly. However disease has spread more rapidly into areas where no disease existed before with the transport and moving of hives. Disease has caused massive losses within the honeybee industry in recent history. Using new technology available to scientists, diseases and parasites can be identified and this information used to prevent damage to hives, the livelihood of many crop farmers and beekeepers around the world. Of these diseases honeybee viruses have become of some concern in recent times. Honeybee viruses’ black queen cell virus (BQCV) and acute bee paralysis virus (ABPV) were found to have genomes consisting of 8550 and 9490 nucleotides respectively. The viruses have two open reading frames (ORFs) which encode a non structural protein at the 5’ ORF and a structural protein at the 3’ ORF. Sacbrood virus (SBV) has a different organisation to BQCV and ABPV where it has a single ORF with the structural genes at the 5’ end and the non structural genes at the 3’ end. In an effort to rapidly identify honeybee viruses a multiplex reverse transcriptase polymerase chain reaction (RT-PCR) was developed Primers were designed within the 3’ open reading frame to amplify fragments of 434bp for SBV, 900bp for ABPV and 316bp for BQCV. RNA was extracted from laboratory infected and naturally infected samples. The PCR products were sequenced and found to be that of the appropriate virus. The primers were tested on naturally infected samples with SBV and BQCV being detected. Another well characterised honeybee virus Kashmir bee virus (KBV) was initially added to the multiplex RT-PCR. However inconsistencies with the multiplex PCR led to the sequencing of a 2 kilobase fragment of the KBV Indian (KBV-in) strain. Three overlapping cDNA fragments of KBV were sequenced and aligned with the full length sequence of KBV and a sequenced capsid region of KBV both from North America. Alignment to ABPV was also completed to observe the homology between KBV-in and ABPV. The KBV-in strain was not highly homologous to the North American strains over the region which was sequenced for KBV-in. ABPV was also not highly homologous over the entire 2 Kb region. However over the region where primers were designed for the RT PCR of KBV, ABPV was highly homologous at 80%. This could have led to the inconsistencies when PCR was done. Primer design and correct strain characterisation is needed before primers are designed to detect more than one virus per reaction. Further characterisation and sequencing of this strain is needed in order to make further comparisons. Propagation methods for honeybee viruses have not changed since these viruses were discovered. There are no suitable cell lines or cell culture techniques available for honeybee viruses. Honeybee viruses have to be manually injected with virus in order for the virus to multiply and be extracted. With the presence of inapparent viruses which could co-infect pupae, a method for pure virus propagations needs to be found. Recombinant baculovirus systems have been used extensively to produce foreign proteins from different viruses using vectors and recombinant technology. In this chapter we inserted the capsid gene from BQCV into a transfer vector under the control of the p10 promoter of Autographa californica. Fractions of the sucrose gradient containing the virus like particles (VLPs) were seen under the electron microscope. A Western blot showed the four capsid proteins at the expected sizes for BQCV capsid. This study therefore has shown that a heterologous system such as baculovirus can be used for virus like particle production. Infectious virus technology has helped gain insight into how viruses work. Using this technology altering honeybee viruses could be used to observe different functionalities of the viruses. An attempt was made to interchange the open reading frames of ABPV and BQCV to observe any changes in virus assembly and infectivity. A fusion PCR strategy was employed to interchange the 5’ and 3’ ORFs of APBV and BQCV. The strategy however was unsuccessful. Alternative strategies could improve the chances of obtaining a chimeric virus.