Chinese Sacbrood Virus Infection Research Articles

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.

Adenosine and L-proline can possibly hinder Chinese Sacbrood virus infection in honey bees via immune modulation

Sacbrood virus (SBV) infects larvae of honey bees, resulting in infected larvae becoming fluid-filled sacs. Our previous studies showed that the extract of herbal medicine, Radix Isatidis, could inhibit Chinese SBV (CSBV) infection in Asian honey bees (Apis cerana). Here, two compounds, adenosine and L-proline, which were previously reported to be associated with immune modulation, were identified in R. Isatidis extract and then selected for an evaluation of their antiviral effect on CSBV infection in A. cerana. Our results revealed that both adenosine and L-proline could significantly mitigate the impact of CSBV on the growth and development of infected larvae and modulate hosts' immune responses by downregulating the expression of immune genes in infected larvae. The results gained from this study suggest that adenosine and L-proline could possibly interfere CSBV infection via immune modulation to avoid exacerbations and nonspecific damage to infected larvae's own tissues.

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.

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

BackgroundSacbrood 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.MethodsHere, 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.ResultsBioassays 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.ConclusionAlthough 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.

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.

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.

Screening of binding proteins that interact with Chinese sacbrood virus VP3 capsid protein in Apis cerana larvae cDNA library by the yeast two-hybrid method

Chinese sacbrood virus (CSBV) causes larval death and apiary collapse of Apis cerana. VP3 is a capsid protein of CSBV but its function is poorly understood. To determine the function of VP3 and screen for novel binding proteins that interact with VP3, we conducted yeast two-hybrid screening, glutathione S-transferase pull-down, and co-immunoprecipitation assays. Galectin (GAL) is a protein involved in immune regulation and host-pathogen interactions. The yeast two-hybrid screen implicated GAL as a major VP3-binding candidate. The assays showed that the VP3 interacted with GAL. Identification of these cellular targets and clarifying their contributions to the host-pathogen interaction may be useful for the development of novel therapeutic and prevention strategies against 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.

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.

The high-throughput production of dsRNA against sacbrood virus for use in the honey bee Apis cerana (Hymenoptera: Apidae).

Sacbrood virus (SBV) is a serious threat to honey bees. Currently, there is no specific drug available for the treatment of SBV that does not affect the quality of the bee product. RNA interference (RNAi) is an important antiviral strategy for disease control. To effectively utilize this technology, the large-scale production and purification of double-stranded RNA (dsRNA) is necessary. Here, a dsRNA-expressing plasmid targeting the VP1 gene of Chinese sacbrood virus (CSBV) was constructed and expressed in Escherichia coli (E. coli) HT115 (DE3). After lysing and ethanol precipitation from E. coli, dsRNA VP1 was purified with an anion exchange chromatography column. Second instar larvae of Apis cerana were fed the purified dsRNA VP1. A significant decrease in larval mortality and the level of expression of the VP1 gene after CSBV infection was demonstrated after the ingestion of dsRNA VP1. This result provides a potential method for the large-scale production of dsRNA to protect A. cerana from CSBV infection.

Differential Gene Transcription in Honeybee (Apis cerana) Larvae Challenged by Chinese Sacbrood Virus (CSBV)

Honey bees are economically important social insect. They are suffering from all kinds of pathogens, especially the virus. In response to pathogens, different immune pathways such as Toll, Imd, Jak-Stat and RNAi are involved. In the present study, the transcription analysis of 32 immune-related genes from Apis cerana challenged by Chinese sacbrood virus (CSBV), the most widely distributed virus in A. cerana, was carried out by qRT-PCR to provide cues for the antiviral mechanism and the effective control of bee viruses. The expression level of 22 genes were statistically changed, including 11 up-regulated genes (catus-2, lys-2, vir, s3a, mta1, faa, vhdl, co-1-iv, ago-1, ago-3, aub) in which 3 (ago-1, ago-3, aub) were related to RNAi pathway, and 11 down-regulated genes (kenny, pgrp-lc, pgrp-s2, abaecin, lys-1, lys-3, domeless, tepa, mlc, dscam, rpl8) related to Toll, Imd, and Jak-Stat pathways. The results indicated CSBV infection in A. cerana may activate a RNA-based antiviral immunity system. This work constituted the first report, under laboratory conditions, about induction of immune related genes in response to CSBV.

TaqMan MGB Probe Fluorescence Real-Time Quantitative PCR for Rapid Detection of Chinese Sacbrood Virus

Sacbrood virus (SBV) is a picorna-like virus that affects honey bees (Apis mellifera) and results in the death of the larvae. Several procedures are available to detect Chinese SBV (CSBV) in clinical samples, but not to estimate the level of CSBV infection. The aim of this study was develop an assay for rapid detection and quantification of this virus. Primers and probes were designed that were specific for CSBV structural protein genes. A TaqMan minor groove binder (MGB) probe-based, fluorescence real-time quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed; specificity was high and there were no cross-reactivity with healthy larvae or other bee viruses. The assay was applied to detect CSBV in 37 clinical samples and its efficiency was compared with clinical diagnosis, electron microscopy observation, and conventional RT-PCR. The TaqMan MGB-based probe fluorescence real-time quantitative PCR for CSBV was more sensitive than other methods tested. This assay was a reliable, fast, and sensitive method that was used successfully to detect CSBV in clinical samples. The technology can provide a useful tool for rapid detection of CSBV. This study has established a useful protocol for CSBV testing, epidemiological investigation, and development of animal models.

Loop-mediated isothermal amplification for rapid detection of Chinese sacbrood virus

Chinese sacbrood virus (CSBV) has emerged as an important etiologic agent of honeybee infections and is lethal for individual bees, even causing the collapse of entire colonies. Although diagnostic methods for CSBV have been established in many clinical laboratories, application of these methods is largely restricted by the apparatus needed to carry out the reaction and by cost, therefore a simpler and less expensive diagnostic method for CSBV infection is required. In this study a simple and inexpensive system is described that is based on the loop-mediated isothermal amplification (LAMP) assay. The LAMP and the polymerase chain reaction (PCR) methods were compared for their ability to detect CSBV in 31 clinical samples, in purified CSBV-LNQY strains or to be able to discriminate between cDNA samples from other viruses. The detection limit of the LAMP method was 1 pg, showing that LAMP is as sensitive as reverse transcriptase (RT)-PCR for CSBV detection. In addition, no DNA band from other related viruses samples was amplified by either method, suggesting that this LAMP assay is as specific as RT-PCR for CSBV detection. All 31 clinical samples that were LAMP assay-positive were also amplified by RT-PCR, however the LAMP assay was faster, more cost effective, and easier to perform as the target gene amplified rapidly, within 2 h, and only a standard laboratory water bath or heat block was required for the reaction. The results demonstrate clearly that this LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of CSBV infection of bees.

Prevention of Chinese Sacbrood Virus Infection in Apis cerana using RNA Interference

Chinese sacbrood virus (CSBV) is the pathogen of Chinese sacbrood disease, which poses a serious threat to honeybee Apis cerana, and tends to cause bee colony and even the whole apiary collapse. Here we report on prevention of CSBV infection by feeding second instar larvae of A. cerana with specific sequences of CSBV double-stranded RNA (dsRNA). Protection of the bee larvae from CSBV by ingestion of CSBV-derived dsRNA was further demonstrated by quantitative real-time PCR (qRT-PCR) and northern blot analysis. The result provides a potential method to protect A. cerana from CSBV infection.