A molecular clone of Chronic Bee Paralysis Virus (CBPV) causes mortality in honey bee pupae (Apis mellifera)

Abstract

Among the many diseases compromising the well-being of the honey bee (Apis mellifera) the chronic paralysis syndrome of adult honey bees is one of the best described. The causative agent, chronic bee paralysis virus (CBPV), is a positive sense, single-stranded RNA virus with a segmented genome. Segment 1 encodes three putative open reading frames (ORFs), including the RNA-dependent RNA polymerase and other non-structural protein coding regions. Segment 2 encodes four putative ORFs, which contain the genes of supposed structural proteins. In this study, we established a reverse genetic system for CBPV by molecular cloning of DNA copies of both genome segments. CBPV rescue was studied in imago and honey bee pupae infection models. Virus replication and progeny virus production was only initiated when capped RNAs of both genome segments were injected in honey bees. As injection of these clonal RNAs caused clinical symptoms similar to wild-type CBPV infection, we conclude that the novel molecular clone fulfilled Koch’s postulates. Our virus clone will enable in-depth analysis of CBPV pathogenesis and help to increase knowledge about this important honey bee disease.

Visualizing Sacbrood Virus of Honey Bees via Transformation and Coupling with Enhanced Green Fluorescent Protein

“…The bicistronic design is a simple method for adding a reporter gene in honey bee viruses. Published honey bee virus clones have mutations that were used as labels detectable only in RT-PCR analyses [10][11][12]. In this study, we inserted 908 bp after the 3 UTR, and the clone was still infectious to host larvae and stable through passages.…”

Bee Viruses: Routes of Infection in Hymenoptera

“…Moreover, crude bee preparations also contain host cellular material that can independently or in synergy with either the inoculated or resident background viruses to influence the virus infection dynamics. An alternative approach would be to synthesize the virus of interest in vitro (Lamp et al, 2016;Ryabov et al, 2019;Seitz et al, 2019;Jin et al, 2020;Yang et al, 2020) thereby ensuring the highest level of purity. Both cell cultures and reverse genetics also allow virus to be produced that is free of contaminants, while reverse genetics also has the option of introducing specific genetic changes to the virus genome (Lamp et al, 2016;Ryabov et al, 2019;Jin et al, 2020).…”

A Reverse Genetics System for the Israeli Acute Paralysis Virus and Chronic Bee Paralysis Virus

“…Lamp et al [34] constructed a deformed wing virus (DWV) infectious clone that was 10,164 bp in length based on the pBR322 vector and identified its infectiousness and pathogenesis. Recently, a molecular clone of CBPV was made based on T vector and confirmed that 5 -cap structures were required for viral replication [35].…”

“…IAPV was close to Chinese strains KX421583 and quite different from the Australia strain (EU436456). We did not perform a phylogenetic analysis on the RdRp of CBPV because Seitz et al 2019 [35] created a phylogenetic tree using the RdRp genes involving our infectious clone CBPV (MF175173 for RNA 1). As shown in their results, current CBPV formed a separate subgroup, which was similar to strains from France, Belgium, and Spain.…”

“…PCR amplification was performed as follows: 30 s at 98 • C, 10 s at 98 • C, 30 s at 61 • C (55 • C for CBPV), 90 s at 72 • C for 35 cycles, and 10 min at 72 • C for an extra extension. The PCR products were subsequently electrophoresed in a 1.5% agarose gel containing GoldView (SBS Genetech Corp. Ltd., Beijing, China); then purified products were cloned into the pEASY-Blunt cloning vector (TransGen Biotech, Beijing, China) and transformed into competent Escherichia coli cells (HB101) for sequencing [35]. For CBPV, two pairs and one pair primers for RNA 1 and RNA 2, respectively, were used to amplify the full sequence of both fragments.…”