Antiviral Activities of a Medicinal Plant Extract Against Sacbrood Virus in Honeybees.

BACKGROUND: Sacbrood is an infectious disease of the honey bee caused by Scbrood virus (SBV) which belongs to the family Iflaviridae and is especially lethal for Asian honeybee Apis cerana. Chinese Sacbrood virus (CSBV) is a geographic strain of SBV. Currently, there is a lack of an effective antiviral agent for controlling CSBV infection in honey bees. METHODS: Here, we explored the antiviral effect of a Chinese medicinal herb Radix isatidis on CSBV infection in A. cerana by inoculating the 3rd instar larvae with purified CSBV and treating the infected bee larvae with R. isatidis extract at the same time. The growth, development, and survival of larvae between the control and treatment groups were compared. The CSBV copy number at the 4th instar, 5th instar, and 6th instar larvae was measured by the absolute quantification PCR method. RESULTS: Bioassays revealed that R. isatidis extract significantly inhibited the replication of CSBV, mitigated the impacts of CSBV on larval growth and development, reduced the mortality of CSBV-infected A. cerana larvae, and modulated the expression of immune transcripts in infected bees. CONCLUSION: Although the mechanism underlying the inhibition of CSBV replication by the medicine plant will require further investigation, this study demonstrated the antiviral activity of R. isatidis extract and provides a potential strategy for controlling SBV infection in honey bees.

Phylogenetic analysis and survey of Apis cerana strain of Sacbrood virus (AcSBV) in Taiwan suggests a recent introduction.

The Sacbrood virus (SBV) is widely distributed in European honey bees, Apis mellifera. AcSBV, a distinct SBV strain in Asian honey bees (A. cerana) causes larva death before pupation and often depopulates colonies, leading to collapse. It is the most severe disease in A. cerana beekeeping. AcSBV infects A. cerana in most natural habitats, yet occurrences were not reported in Taiwan before 2015 and were not a concern for local beekeepers. However, in 2016, A. cerana beekeepers in central Taiwan reported SBV-like symptoms. We screened samples of larvae using RT-PCR and surveyed asymptomatic apiaries in north Taiwan. Phylogenetic analyses suggested that AcSBV isolates from central Taiwan were introduced; all isolates had high similarity in sequences to AcSBV genomes identified in mainland China, Vietnam, and Korea and distinct differences to SBV sequence identified in Taiwan. The overall prevalence in symptomatic colonies was low. No latent infections were detected in asymptomatic colonies. The AcSBV epizootic may not yet have reached its highest potential.

Chinese Sacbrood virus infection in Asian honey bees (Apis cerana cerana) and host immune responses to the virus infection.

Chinese Sacbrood virus (CSBV) is a positive-stranded RNAvirus that infects both the European honey bee (Apis mellifera) and the Asian honey bee (A. cerana). However, CSBV has much more devastating effects on Asian honey bees than on European honey bees, posing a serious threat to the agricultural and natural ecosystems that rely on A. cerana for pollination service. Using quantitative RT-PCR method, we conducted studies to examine the CSBV infection in Asian honey bee colonies and immune responses of individual bees in response to CSBV infection. Our study showed that CSBV could cause infection in different developmental stages of workers including eggs, larvae, pupae, newly emerged workers, and foraging workers. In addition, evaluating the tissue tropism and transmission of CSBV in infected bees showed that CSBV was detected in the ovaries, spermatheca, and feces of queens as well as semen of drones of the same colonies, suggesting an existence of vertical transmission of CSBV in Asian honey bees. Further, the detection of CSBV in colony food suggests that healthy bees could pick the infection by the virus-contaminated food, and therefore, a possible existence of a food-borne transmission pathway of CSBV in Asian bee colonies. The expression analysis of transcripts (defensin, abaecin, apidaecin, and hymenoptaecin) involving innate antiviral immune pathways showed that CSBV infection could induce significant immune responses in infected bees. However, the immune responses to CSBV infection varied among different development stages with eggs exhibiting the lowest level of immune expression and forager workers exhibiting the highest level of immune gene expression. The results obtained in the study yield important insights into the mechanisms underlying disease pathogenesis of CSBV infections in Asian honey bees and provide valuable information for a rational design of disease control measures.

Enhancement of antiviral activity of egg yolk antibodies against Chinese sacbrood virus.

Chinese sacbrood virus (CSBV) poses a serious threat to the apiculture of China. Although several approaches have been attempted to control CSBV infection, their applications have been greatly limited in practical breeding of honeybees due to poor effectiveness. Egg yolk antibodies (EYA) have shown a promising protection for bees against CSBV infection. This study was conducted to produce high titer EYA and then further improve their antiviral effect. Among three vaccination groups, the EYA titer in graphene oxide-chitosan group was highest (1.591 ± 0.145), in Freund's group was modest (1.195 ± 0.040), and in white oil group was lowest (1.058 ± 0.056). After three injections of each vaccine in hens, EYA were produced at the highest level with a 14-day period. After application of EYA for more than two years in actual bee breeding, prevention and treatment assays showed that EYA confered 98.9 to 100% protection from CSBV infection. The mortality of the control group reached to a range of 91.2 to 100%. This study demonstrated that the high titer EYA have been successfully prepared with significant anti-CSBV activity and that these antibodies may feasibly be used for CSBV treatment to meet the practical needs of apiculture.

Lipidomic Profiling Reveals Distinct Differences in Sphingolipids Metabolic Pathway between Healthy Apis cerana cerana larvae and Chinese Sacbrood Disease.

Chinese sacbrood disease (CSD), which is caused by Chinese sacbrood virus (CSBV), is a major viral disease in Apis cerana cerana larvae. Analysis of lipid composition is critical to the study of CSBV replication. The host lipidome profiling during CSBV infection has not been conducted. This paper identified the lipidome of the CSBV-larvae interaction through high-resolution mass spectrometry. A total of 2164 lipids were detected and divided into 20 categories. Comparison of lipidome between healthy and CSBV infected-larvae showed that 266 lipid species were altered by CSBV infection. Furthermore, qRT-PCR showed that various sphingolipid enzymes and the contents of sphingolipids in the larvae were increased, indicating that sphingolipids may be important for CSBV infection. Importantly, Cer (d14:1 + hO/21:0 + O), DG (41:0e), PE (18:0e/18:3), SM (d20:0/19:1), SM (d37:1), TG (16:0/18:1/18:3), TG (18:1/20:4/21:0) and TG (43:7) were significantly altered in both CSBV_24 h vs. CK_24 h and CSBV_48 h vs. CK_48 h. Moreover, TG (39:6), which was increased by more than 10-fold, could be used as a biomarker for the early detection of CSD. This study provides evidence that global lipidome homeostasis in A. c. cerana larvae is remodeled after CSBV infection. Detailed studies in the future may improve the understanding of the relationship between the sphingolipid pathway and CSBV replication.

Transcriptome Profiling Reveals a Novel Mechanism of Antiviral Immunity Upon Sacbrood Virus Infection in Honey Bee Larvae (Apis cerana).

The honey bee is one of the most important pollinators in the agricultural system and is responsible for pollinating a third of all food we eat. Sacbrood virus (SBV) is a member of the virus family Iflaviridae and affects honey bee larvae and causes particularly devastating disease in the Asian honey bees, Apis cerana. Chinese Sacbrood virus (CSBV) is a geographic strain of SBV identified in China and has resulted in mass death of honey bees in China in recent years. However, the molecular mechanism underlying SBV infection in the Asian honey bee has remained unelucidated. In this present study, we employed high throughput next-generation sequencing technology to study the host transcriptional responses to CSBV infection in A. cerana larvae, and were able to identify genome-wide differentially expressed genes associated with the viral infection. Our study identified 2,534 differentially expressed genes (DEGs) involved in host innate immunity including Toll and immune deficiency (IMD) pathways, RNA interference (RNAi) pathway, endocytosis, etc. Notably, the expression of genes encoding antimicrobial peptides (abaecin, apidaecin, hymenoptaecin, and defensin) and core components of RNAi such as Dicer-like and Ago2 were found to be significantly upregulated in CSBV infected larvae. Most importantly, the expression of Sirtuin target genes, a family of signaling proteins involved in metabolic regulation, apoptosis, and intracellular signaling was found to be changed, providing the first evidence of the involvement of Sirtuin signaling pathway in insects' immune response to a virus infection. The results obtained from this study provide novel insights into the molecular mechanism and immune responses involved in CSBV infection, which in turn will contribute to the development of diagnostics and treatment for the diseases in honey bees.

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

Sacbrood virus (SBV) of honey bees is a picornavirus in the genus Iflavirus. Given its relatively small and simple genome structure, single positive-strand RNA with only one ORF, cloning the full genomic sequence is not difficult. However, adding nonsynonymous mutations to the bee iflavirus clone is difficult because of the lack of information about the viral protein processes. Furthermore, the addition of a reporter gene to the clones has never been accomplished. In preliminary trials, we found that the site between 3' untranslated region (UTR) and poly(A) can retain added sequences. We added enhanced green fluorescent protein (EGFP) expression at this site, creating a SBV clone with an expression tag that does not affect virus genes. An intergenic region internal ribosome entry site (IRES) from Black queen cell virus (BQCV) was inserted to initiate EGFP expression. The SBV-IRES-EGFP clone successfully infected Apis cerana and Apis mellifera, and in A. cerana larvae, it was isolated and passaged using oral inoculation. The inoculated larvae had higher mortality and the dead larvae showed sacbrood symptoms. The added IRES-EGFP remained in the clone through multiple passages and expressed the expected EGFP in all infected bees. We demonstrated the ability to add gene sequences in the site between 3'-UTR and poly(A) in SBV and the potential to do so in other bee iflaviruses; however, further investigations of the mechanisms are needed. A clone with a desired protein expression reporter will be a valuable tool in bee virus studies.

Identification of Immune Response to Sacbrood Virus Infection in Apis cerana Under Natural Condition.

Chinese sacbrood virus (CSBV) is a serious threat to eastern honeybees (Apis cerana), especially larvae. However, the pathological mechanism of this deadly disease remains unclear. Here, we employed mRNA and small RNA (sRNA) transcriptome approach to investigate the microRNAs (miRNAs) and small interfering RNAs (siRNAs) expression changes of A. cerana larvae infected with CSBV under natural condition. We found that serine proteases involved in immune response were down-regulated, while the expression of siRNAs targeted to serine proteases were up-regulated. In addition, CSBV infection also affected the expression of larvae cuticle proteins such as larval cuticle proteins A1A and A3A, resulting in increased susceptibility to CSBV infection. Together, our results provide insights into sRNAs that they are likely to be involved in regulating honeybee immune response.

Genetic and phylogenetic analysis of Chinese sacbrood virus isolates from Apis mellifera.

BACKGROUND: Sacbrood virus (SBV) is one of the most pathogenic honeybee viruses that exhibits host specificity and regional variations. The SBV strains that infect the Chinese honeybee Apis cerana are called Chinese SBVs (CSBVs). METHODS: In this study, a CSBV strain named AmCSBV-SDLY-2016 (GenBank accession No. MG733283) infecting A. mellifera was identified by electron microscopy, its protein composition was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and agar gel immunodiffusion assay, and its nucleotide sequence was identified using a series of reverse-transcription polymerase chain reaction fragments of AmCSBV-SDLY-2016 generated using SBV/CSBV-specific primers. To investigate phylogenetic relationships of the CSBV isolates, a phylogenetic tree of the complete open reading frames (ORF) of the CSBV sequences was constructed using MEGA 6.0; then, the similarity and recombination events among the isolated CSBV strains were analyzed using SimPlot and RDP4 software, respectively. RESULTS: Sequencing results revealed the complete 8,794-nucleotide long complete genomic RNA of the strain, with a single large ORF (189-8,717) encoding 2,843 amino acids. Comparison of the deduced amino acid sequence with the SBV/CSBV reference sequences deposited in the GenBank database identified helicase, protease, and RNA-dependent RNA polymerase domains; the structural genes were located at the 5' end, whereas the non-structural genes were found at the 3' end. Multiple sequence alignment showed that AmCSBV-SDLY-2016 had a 17-amino acid (aa) and a single aa deletion at positions 711-729 and 2,128, respectively, as compared with CSBV-GD-2002, and a 16-aa deletion (positions 711-713 and 715-728) as compared with AmSBV-UK-2000. However, AmCSBV-SDLY-2016 was similar to the CSBV-JLCBS-2014 strain, which infects A. cerana. AmCSBV-SDLY-2016 ORF shared 92.4-97.1% identity with the genomes of other CSBV strains (94.5-97.7% identity for deduced amino acids). AmCSBV-SDLY-2016 was least similar (89.5-90.4% identity) to other SBVs but showed maximum similarity with the previously reported CSBV-FZ-2014 strain. The phylogenetic tree constructed from AmCSBV-SDLY-2016 and 43 previously reported SBV/CSBV sequences indicated that SBV/CSBV strains clustered according to the host species and country of origin; AmCSBV-SDLY-2016 clustered with other previously reported Chinese and Asian strains (AC genotype SBV, as these strains originated from A. cerana) but was separate from the SBV genomes originating from Europe (AM genotype SBV, originating from A. mellifera). A SimPlot graph of SBV genomes confirmed the high variability, especially between the AC genotype SBV and AM genotype SBV. This genomic diversity may reflect the adaptation of SBV to specific hosts, ability of CSBV to cross the species barrier, and the spatial distances that separate CSBVs from other SBVs.

Identification of the Novel Host Protein Interacting With the Structural Protein VP1 of Chinese Sacbrood Virus by Yeast Two-Hybrid Screening.

Chinese sacbrood virus (CSBV) is the major cause and lead to the collapse of Apis cerana colonies. VP1, the structural protein of CSBV, shows the highest variation in the amino acid sequences among proteins from different CSBV strains as well as exhibits excellent immunogenicity. However, its function with host protein still remains unclear. To clarify its function with host protein, we screened out host cellular proteins that interact with VP1 using the membrane protein yeast two-hybrid system. In addition, we verified interactions between heat shock protein 70 cognate 5 (Hsp70-c5) and VP1 using glutathione S-transferase (GST) pull-down and co-immunoprecipitation assays. VP1 and Hsp70-c5 were colocalized in the cytoplasm and nucleus. Using western blot and real-time polymerase chain reaction (PCR), Hsp70-c5 expression in CSBV-infected larvae was upregulated compared with that in healthy larvae. We observed that when we silenced Hsp70-c5, VP1 expression was significantly downregulated. These results demonstrate that Hsp70-c5 is involved in at least one stage(s) of the viral life cycle.

Purification of Chinese Sacbrood Virus (CSBV), Gene Cloning and Prokaryotic Expression of its Structural Protein VP1.

The aim of this study was to purify the Chinese Sacbrood Virus Beijing Miyun (BJMY-CSBV) from infected Apis cerana larvae, clone structural protein gene VP1 (named BJMY-CSBV-VP1), and investigate its biological information. The result indicated that the capsid of CSBV is of spherical shape. Gene clone experiment showed that the BJMY-CSBV-VP1 gene sequence comprised 945 bp, encoding 315 amino acids with relative molecular weight of 35.59 kDa and isoelectric point 9.38 pI. Phylogenetic analysis of amino acid sequences showed that the BJMY-CSBV-VP1 and LNDD_2015 were grouped together. Protein secondary structure prediction showed that the gene contained two α-helices, thirteen ß-folds, six polypeptide binding sites, and no disulfide bridge. Simultaneously, the BJMY-CSBV-VP1 was ligated to the expression vector pET32a(+) and then transformed into the Escherichia coli BL21 (DE3) for prokaryotic expression. The optimal expression experiment revealed that the protein was found in the inclusion body. The recombinant protein was successfully purified by washing buffer combined with supersonic fragmentation. In this study, we obtained the purified BJMY-CSBV particles, cloned BJMY-CSBV-VP1 gene, investigated the detailed information of the gene by analyzing the sequence, and obtained the purified recombinant protein, which could help for further understanding of the function of the structural protein gene VP1.

Preparation and Application of Egg Yolk Antibodies Against Chinese Sacbrood Virus Infection.

Chinese sacbrood virus (CSBV) infects Apis cerana larvae, resulting in the inability of the larvae to pupate and their consequent death, which may pose a serious threat to entire colonies. As there is no effective medical treatment for CSBV infections, further studies are necessary. In this study, an effective treatment for CSBV is described, based on a specific immunoglobulin Y (IgY) from egg yolk against CSBV. The inactivated vaccine was produced by ultracentrifugation and formalin treatment, using CSBV purified from a natural outbreak. The specific IgY was produced by immunization of white leghorn hens with the vaccine. An enzyme-linked immunosorbent assay using purified CSBV as the coating antigen revealed that the anti-CSBV IgY titer began increasing in the egg yolk on the 14th day post-immunization, reaching a peak on day 42, and anti-CSBV IgY remained at a high level until day 91. IgY isolated from the combinations of egg yolk collected between days 42-91 was purified by PEG and ammonium sulfate precipitation. In three repeated protection experiments using A. cerana larvae inoculated with CSBV, the survival rate of larvae was more than 80%, and the titer of anti-CSBV IgY was more than 25 and 24 when the larvae were fed IgY 24 h after and before inoculation with CSBV, respectively. Therefore, 400 colonies infected with CSBV were treated by feeding sugar containing IgY solutions with an antibody titer of 25, and the cure rate was 95-100%. Three hundred susceptible colonies were protected by feeding the larvae with sugar containing IgY solutions with an antibody titer of 24, and the protection rate was 97%. The results clearly suggest that a specific IgY was obtained from hens immunized with an inactivated-CSBV vaccine; this may be a novel method for controlling CSBV infection.

Chinese sacbrood virus infection in Apis mellifera, Shandong, China, 2016.

The Chinese sacbrood virus (CSBV) was first isolated from Apis cerana in 1972. However, the biological characteristics of the CSBV that naturally infects Apis mellifera, causing larval death, have not been reported yet. In the present study, natural CSBV infection was evaluated using clinical symptoms of A. mellifera larvae, RT-PCR, electron microscopy, agar gel immunodiffusion assays, and virus analysis in inoculated A. cerana larvae. The isolated CSBV strain was named AmCSBV-SDLY-2016. Subsequently, AmCSBV-SDLY-2016 was analyzed by constructing a phylogenetic tree using VP1. Data from the phylogenetic tree suggested that AmCSBV-SDLY-2016 is evolutionarily close to JLCBS-2014. It was also observed that CSBV crossed the species barrier, causing the death of A. mellifera larvae.