Sacbrood Virus Research Articles

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

A honeybee colony, part of an apiary of nine, showed abnormalities in brood pattern and was thus presented for study. A classic veterinary medicine approach has allowed the diagnosis of a severe case of sacbrood virus (SBV) confirmed by a high viral load in affected larvae. SBV is known to infect larvae of the honeybee (Apis mellifera), resulting in failure to pupate and ultimately death of infected larvae. Several contributing factors combined, among them the parasite Varroa destructor, have been identified in this particular affected colony to explain the clinical outbreak of the disease whereas, in the majority of cases, infected colonies remain asymptomatic. As no specific cure of honeybee viruses is available, the management of these contributing factors is essential, including feeding of colonies and control of the Varroa parasite. After implementation of management solutions, the colony rapidly recovered in six weeks, but did not recommence honey production and remained at higher risk of a winter collapse. An earlier control management would have been more effective: regular visits of the colonies by the beekeepers should be the rule in order to detect abnormalities and also to detect and eliminate as early as possible the combination of factors that contribute to the proliferation of the virus.

Codon Optimization, Expression in Escherichia coli, and Immunogenicity of Recombinant Chinese Sacbrood Virus (CSBV) Structural Proteins VP1, VP2, and VP3.

Chinese sacbrood virus (CSBV) is a small RNA virus family belonging to the genus Iflavirus that causes larval death, and even the collapse of entire bee colonies. The virus particle is spherical, non-enveloped, and its viral capsid is composed of four proteins, although the functions of the structural proteins are unclear. In this study, we used codon recoding to express the recombinant proteins VP1, VP2, and VP3 in Escherichia coli. SDS-PAGE analysis and Western blotting revealed that the target genes were expressed at high levels. Mice were then immunized with the purified, recombinant proteins, and antibody levels and lymphocyte proliferation were analyzed by ELISA and the MTT assay, respectively. The results show that the recombinant proteins induced high antibody levels and promoted lymphocyte proliferation. Polyclonal antibodies directed against these proteins will aid future studies of the molecular pathogenesis of CSBV.

Virus Status, Varroa Levels, and Survival of 20 Managed Honey Bee Colonies Monitored in Luxembourg Between the Summer of 2011 and the Spring of 2013

Abstract Twenty managed honey bee colonies, split between 5 apiaries with 4 hives each, were monitored between the summer of 2011 and spring of 2013. Living bees were sampled in July 2011, July 2012, and August 2012. Twenty-five, medium-aged bees, free of varroa mites, were pooled per colony and date, to form one sample. Unlike in France and Belgium, Chronic Bee Paralysis Virus (CBPV) has not been found in Luxembourg. Slow Bee Paralysis Virus (SBPV) and Israeli Acute Paralysis Virus (IAPV) levels were below detection limits. Traces of Kashmir Bee Virus (KBV) were amplified. Black Queen Cell Virus (BQCV), Varroa destructor Virus-1 (VDV-1), and SacBrood Virus (SBV) were detected in all samples and are reported from Luxembourg for the first time. Varroa destructor Macula- Like Virus (VdMLV), Deformed Wing Virus (DWV), and Acute Bee Paralysis Virus (ABPV) were detected at all locations, and in most but not all samples. There was a significant increase in VDV-1 and DWV levels within the observation period. A principal component analysis was unable to separate the bees of colonies that survived the following winter from bees that died, based on their virus contents in summer. The number of dead varroa mites found below colonies was elevated in colonies that died in the following winter. Significant positive relationships were found between the log-transformed virus levels of the bees and the log-transformed number of mites found below the colonies per week, for VDV-1 and DWV. Sacbrood virus levels were independent of varroa levels, suggesting a neutral or competitive relationship between this virus and varroa.

Thai sacbrood and sacbrood viruses versus European foulbrood of hive bees in India – a review

Thai sacbrood virus (TSBV) disease has killed 95% of Apis cerana colonies in India, while sacbrood virus (SBV) disease has affected only 15% of Apis mellifera colonies. The symptoms are sometimes confused with those of the bacterial disease European foulbrood (EFB) in A. cerana. TSBV and SBV are species specific, but closely related strains. Both are isometric in shape, 30 nm in diameter, and are unstable. They have good antigenecity (1:200 titer). Sequencing TSBV and SBV genomes yields fragments of 773 and 822 nucleotide lengths, respectively. The respective sequences have been submitted to NCBI, USA GenBank database under accession numbers EU 156753 and 232663. Phylogenetically, TSBV forms a different group from SBV. In spite of these studies, confusion regarding to the identification of TSBV still prevails in the southern part of India.

Potential for virus transfer between the honey bees Apis mellifera and A. cerana

Viruses seem to play a key role in European honey bee, Apis mellifera health, and have a much broader host spectrum than previously thought. Few studies have investigated interspecific virus transfer within the genus Apis. The introduction of A. mellifera into Asia exposed endemic Apis species to the risk of obtaining new viruses or viral strains and vice versa. To investigate the potential for host shifts, virus prevalence and sequences were monitored over three years in single and mixed-species apiaries hosting introduced A. mellifera and endemic Apis cerana. Deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), and sacbrood virus (SBV) were found, but not KBV, VDV-1, ABPV, or CBPV. Virus infections and prevalence were generally lower in A. cerana compared to A. mellifera, and varied over the years. The sequence data provided evidence for interspecific transfer of IAPV, BQCV, and DWV, but SBV strains seem to be species specific. Prevalence and sequence results taken together indicate that interspecific transfers of viruses are rare, even if honey bees are kept in close proximity. We discuss the pattern observed in the context host specificity and resistance. Our understanding of the extent of these exchanges is limited by a lack of knowledge on the mechanisms of adaptation of viruses to different hosts.

Occurrence, detection, and quantification of economically important viruses in healthy and unhealthy honey bee (Hymenoptera: Apidae) colonies in Canada

AbstractThe occurrence, quantification, and distribution patterns of deformed wing virus (DWV) and sacbrood virus (SBV), (family Iflaviridae); black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), and acute bee paralysis virus (ABPV) (family Dicistroviridae), and chronic bee paralysis virus (CBPV) (unclassified), were characterised in 80 “healthy” honey bee (Apis mellifera Linnaeus; Hymenoptera: Apidae) colonies and 23 “unhealthy” colonies by employing reverse transcription polymerase chain reaction (RT-PCR) for virus identification and quantitative real-time polymerase chain reaction (qPCR) for quantification. All seven viruses were common but the most prevalent viruses were DWV, followed by BQCV and IAPV. For most viruses, prevalence in surviving but unhealthy colonies in spring did not differ from that of healthy baseline colony levels in fall suggesting spring prevalence level would not be a useful metric for diagnosis of factors contributing to colony loss. Sacbrood virus was the only virus that was more prevalent in unhealthy colonies from Manitoba, Canada than in healthy from colonies across Canada but did not differ from healthy colonies within Manitoba. Multiple infections were ubiquitous with a few colonies having simultaneous infection with as many as five viruses. Among the three viruses quantified by qPCR, DWV had the highest relative concentrations in pooled samples of worker bees. Deformed wing virus was the only virus within healthy colonies that differed in fall concentration among provinces and was at high levels in unhealthy colonies. Black queen cell virus was positively correlated with IAPV across all samples. Our study provides the first major baseline study of viruses in Canadian honey bees.

Differential Expression of Immune-Related Genes in Larval and Adult Apis mellifera Honeybees Challenged with Korean Sacbrood Virus

Recently, the Korean sacbrood virus (KSBV) caused great loss of honeybee colonies in Korean Apis mellifera. Three antimicrobial peptide genes (AMPs) (abaecin (ABA), defensin (DEF) and hymenoptaecin (HYM)) and two immune-related enzyme genes (phenol-oxidase (PO) and lysozyme (LYS)) were investigated using qRT-PCR as a measure of the defense reactions of honeybee larvae and adults challenged with KSBV in vitro. Transcript levels of three AMPs were upregulated in adults exposed to KSBV, compared to control adults. ABA and DEF transcripts were 22.7 and 21.9 fold higher (P<0.05), respectively, after 24h exposure while HYM transcript did not change significantly. At this time point, PO and LYS mRNA levels were 18.08 and 16.03 fold higher, respectively, in KSBVinfected adult bees compared to control adults (P<0.05). By contrast, the larvae infected with KSBV had downregulated expression of AMPs and slight up-regulation of PO and LYS compared to the controls. These results suggest that the differential expression of the genes studied could indicate their involvement in defense processes against KSBV in adult and larva bees.

전북지역 토종벌에서 낭충봉아부패병 관찰

This study was carried out to investigate the Sacbrood virus (SBV) of Korean native honeybees causing serious damage in Jeonbuk area. Korean native honeybees completing the after overwinter 60 farms and March to April active phase adult bees and larvae 52 farms were collected from farms in 7 counties. Active phase of the adult bees 39 (75.0%) and larvae 24 (46.2%) farms was infected with SBV in 52 farms. This result indicate that SBV was the highest in Imsil-gun than other areas.

Characterization of gut bacteria at different developmental stages of Asian honey bees, Apis cerana

Previous surveys have shown that adult workers of the Asian honey bee Apis cerana harbor four major gut microbes (Bifidobacterium, Snodgrassella alvi, Gilliamella apicola, and Lactobacillus). Using quantitative PCR we characterized gut bacterial communities across the life cycle of A. cerana from larvae to workers. Our results indicate that the presence and quantity of these four bacteria were low on day 1, increased rapidly after day 5, and then peaked during days 10–20. They stabilized from days 20–25 or days 25–30, then dropped to a low level at day 30. In addition, the larvae infected by Sacbrood virus or European foulbrood had significantly lower copies of 16S rRNA genes than healthy individuals.

Metatranscriptomic analyses of honey bee colonies.

Honey bees face numerous biotic threats from viruses to bacteria, fungi, protists, and mites. Here we describe a thorough analysis of microbes harbored by worker honey bees collected from field colonies in geographically distinct regions of Turkey. Turkey is one of the World's most important centers of apiculture, harboring five subspecies of Apis mellifera L., approximately 20% of the honey bee subspecies in the world. We use deep ILLUMINA-based RNA sequencing to capture RNA species for the honey bee and a sampling of all non-endogenous species carried by bees. After trimming and mapping these reads to the honey bee genome, approximately 10% of the sequences (9–10 million reads per library) remained. These were then mapped to a curated set of public sequences containing ca. Sixty megabase-pairs of sequence representing known microbial species associated with honey bees. Levels of key honey bee pathogens were confirmed using quantitative PCR screens. We contrast microbial matches across different sites in Turkey, showing new country recordings of Lake Sinai virus, two Spiroplasma bacterium species, symbionts Candidatus Schmidhempelia bombi, Frischella perrara, Snodgrassella alvi, Gilliamella apicola, Lactobacillus spp.), neogregarines, and a trypanosome species. By using metagenomic analysis, this study also reveals deep molecular evidence for the presence of bacterial pathogens (Melissococcus plutonius, Paenibacillus larvae), Varroa destructor-1 virus, Sacbrood virus, and fungi. Despite this effort we did not detect KBV, SBPV, Tobacco ringspot virus, VdMLV (Varroa Macula like virus), Acarapis spp., Tropilaeleps spp. and Apocephalus (phorid fly). We discuss possible impacts of management practices and honey bee subspecies on microbial retinues. The described workflow and curated microbial database will be generally useful for microbial surveys of healthy and declining honey bees.

Lower virus infections in Varroa destructor-infested and uninfested brood and adult honey bees (Apis mellifera) of a low mite population growth colony compared to a high mite population growth colony.

A comparison was made of the prevalence and relative quantification of deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV) and sac brood virus (SBV) in brood and adult honey bees (Apis mellifera) from colonies selected for high (HMP) and low (LMP) Varroa destructor mite population growth. Two viruses, ABPV and SBV, were never detected. For adults without mite infestation, DWV, IAPV, BQCV and KBV were detected in the HMP colony; however, only BQCV was detected in the LMP colony but at similar levels as in the HMP colony. With mite infestation, the four viruses were detected in adults of the HMP colony but all at higher amounts than in the LMP colony. For brood without mite infestation, DWV and IAPV were detected in the HMP colony, but no viruses were detected in the LMP colony. With mite infestation of brood, the four viruses were detected in the HMP colony, but only DWV and IAPV were detected and at lower amounts in the LMP colony. An epidemiological explanation for these results is that pre-experiment differences in virus presence and levels existed between the HMP and LMP colonies. It is also possible that low V. destructor population growth in the LMP colony resulted in the bees being less exposed to the mite and thus less likely to have virus infections. LMP and HMP bees may have also differed in susceptibility to virus infection.

Complete genome of Chinese sacbrood virus from Apis cerana and analysis of the 3C-like cysteine protease.

The complete genome sequence of Chinese sacbrood virus (CSBV), isolated from diseased larvae of Apis cerana in Fujian province, China was analyzed. The viral genome consisted of 8,800 nucleotides, encoding 2,848 amino acids. A phylogenetic tree analysis showed the sacbrood virus (SBV) segregated into three distinct groups. The isolates originated from A. c. indica and were the first distinct evolutionary group. The AcSBV-SBM2 isolate, from A. c. cerana, belonged to the second distinct group. The remaining SBV isolates formed the third group. The phylogenetic relationships of SBV isolates suggest that they are derived from similar honeybee species or geographic origins. The 3C-like cysteine protease protein plays an important role in viral replication. The 3C-like cysteine protease protein of CSBV-FZ was predicted to contain a transmembrane domain. The subcellular localization of 3C-like cysteine protease was distributed as discrete punctate inclusions and co-localized with VP1 of CSBV. These results suggest that the non-structural protein 3C-like cysteine protease might be involved in viral replication. Insect cell cultures can further advance our understanding of picorna-like virus replication.

Prevalence of Four Common Bee RNA Viruses in Eastern Bee Populations in Yunnan Province, China

Deformed wing virus (DWV), black queen cell virus (BQCV), sacbrood virus (SBV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV) and chronic bee paralysis virus (CBPV) are the most common RNA viruses in bee population worldwide. To determine the prevalence of these viruses in Apis cerana in Yunnan Province, China, 270 adult Eastern worker bee (A. cerana) samples have been collected from nine major bee breeding regions in Yunnan province for screening. Our data showed that BQCV, DWV, KBV and SBV are widely prevalent in Yunnan Province. The total prevalence rates of KBV, DWV, SBV and BQCV in Yunnan province were 3%, 14.8%, 24.1% and 36.3%, respectively. BQCV was detected in all selected regions except Jinghong City. The prevalence of KBV in Tengchong County is the first evidence, to our knowledge, showing that Eastern bees could be infected by KBV. The highest infection rate of SBV was found in Kunming city (56.7%). Sequence analysis demonstrated high nucleotide homology between the isolated BQCV, DWV, KBV and SBV strains. Phylogenetic analysis showed that except SBV (YNO7), Yunnan isolates can be likely clustered into independent branches, which indicates the possibility of geographic origins.

Efficacy of silver ions against Sacbrood virus infection in the Eastern honey bee Apis cerana.

Although silver is known to be a broad-spectrum biocidal agent, the effects of this metal against Sacbrood virus have not yet been investigated. In this study, we evaluated the efficacy of silver ions against natural Korean sacbrood virus (KSBV) infection of Apis (A.) cerana. Ten KSBV-infected colonies containing A. cerana with similar strength and activity were selected from an apiary located in Bosung-gun (Korea). Among these, five colonies were randomly assigned to the treatment group that was fed sugar syrup containing 0.2 mg/L silver ions. The other colonies were assigned to the untreated control group in which bees were given syrup without the silver ions. To assess the efficacy of the silver ions, colony strength, colony activity, and the number of dead larvae per hive were measured. During the experimental period, the test group maintained its strength and activity until day 32 while those of bees in the control group decreased sharply after day 8 to 16. Survival duration of the test group was significantly longer (40 days) than that of the control group (21 days). These results strongly indicated that silver ions are effective against KSBV infection in A. cerana.

Interactions of Tropilaelaps mercedesae, honey bee viruses and immune response in Apis mellifera

Tropilaelaps mites are the major health threat to Apis mellifera colonies in Asia because of their widespread occurrence, rapid population growth and potential ability to transfer bee viruses. Honey bee immune responses in the presence of feeding mites may occur in response to mite feeding, to the presence of viruses, or to both. In this study, the mRNA expression levels were measured for three antimicrobial peptide encoding genes (abaecin, apidaecin and hymenoptaecin) and a phagocytosis receptor gene (eater) in worker brood infested with different numbers of actively feeding T. mercedesae. Also, all samples were measured for the amount of acute bee paralysis virus (ABPV), black queen cell virus (BQCV), deformed wing virus (DWV), Kashmir bee virus (KBV) and sacbrood virus (SBV). Using an artificial mite inoculation protocol, the analysis showed that apidaecin was significantly down-regulated when tan-bodied pupae were infested with 1–2 mites and when capping of the cells of newly sealed larvae were opened and closed without mite inoculation (o/c) as compared to the control group (undisturbed brood, no mite inoculation). Reduced transcription levels of the eater gene were also recorded in the o/c group. However, an up-regulation of apidaecin and eater genes was observed in highly infested pupae when compared to o/c group. This occurrence is perhaps due to an adaptive response of the bees to higher mite infestations by up-regulating their immune expression. No significant expression differences were detected for abaecin and hymenoptaecin and the viruses ABPV, KBV and SBV were not detected. However, 86.7% of the pupae were infected with DWV, 83.3% were infected with BQCV and 73% were infected by both of these viruses. In addition, the Tropilaelaps-inoculated pupae showed higher levels and incidence of DWV compared to uninfested pupae. The presence of these two honey bee viruses was not related to the number of T. mercedesae infesting the pupae. Also, the presence of variable levels of DWV and low levels of BQCV did not provoke any expression differences for any of the targeted genes. Overall, this research indicates that feeding by Tropilaelaps mites produces an immune response, that the level of viruses did not produce a correlated immune response by the four genes tested and that Tropilaelaps may be a potential vector of DWV but not to a high degree. The data indicated that the major impact of Tropilaelaps infestation is caused by the mite itself.

Vertical transmission of honey bee viruses in a Belgian queen breeding program.

BackgroundThe Member States of European Union are encouraged to improve the general conditions for the production and marketing of apicultural products. In Belgium, programmes on the restocking of honey bee hives have run for many years. Overall, the success ratio of this queen breeding programme has been only around 50%. To tackle this low efficacy, we organized sanitary controls of the breeding queens in 2012 and 2014.ResultsWe found a high quantity of viruses, with more than 75% of the egg samples being infected with at least one virus. The most abundant viruses were Deformed Wing Virus and Sacbrood Virus (≥40%), although Lake Sinai Virus and Acute Bee Paralysis Virus were also occasionally detected (between 10-30%). In addition, Aphid Lethal Paralysis Virus strain Brookings, Black Queen Cell Virus, Chronic Bee Paralysis Virus and Varroa destructor Macula-like Virus occurred at very low prevalences (≤5%). Remarkably, we found Apis mellifera carnica bees to be less infected with Deformed Wing Virus than Buckfast bees (p < 0.01), and also found them to have a lower average total number of infecting viruses (p < 0.001). This is a significant finding, given that Deformed Wing Virus has earlier been shown to be a contributory factor to winter mortality and Colony Collapse Disorder. Moreover, negative-strand detection of Sacbrood Virus in eggs was demonstrated for the first time.ConclusionsHigh pathogen loads were observed in this sanitary control program. We documented for the first time vertical transmission of some viruses, as well as significant differences between two honey bee races in being affected by Deformed Wing Virus. Nevertheless, we could not demonstrate a correlation between the presence of viruses and queen breeding efficacies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0386-9) contains supplementary material, which is available to authorized users.

Derivation of cell-adapted Sacbrood virus (SBV) from the native Korean honeybee

Sacbrood virus (SBV), a causative agent of larval death in honeybees, is one of the most devastating diseases in bee industry throughout the world. Lately the Korean Sacbrood virus (KSBV) induced great losses in Korean honeybee (Apis cerana) colonies. However, there is no culture system available for honeybee viruses, including SBV, therefore, the research on honeybee viruses is practically limited until present. In this study, we investigated the growth and replication of SBV in cell cultures. The replication signs of KSBV after passages from mammalian cells was identified and confirmed by using combined approaches with nested, quantitative, negative-strand PCR and electron microscopy along with in vivo experiment. The results revealed that mammalian cell lines, including Vero cells could support the replication KSBV. Although there were no signs of cytopathic effect (CPE) in cells, it was for the first time demonstrated that SBV could be replicated in cells through the sequential passages linked with cell adaptation. KSBV from the present study would be a valuable source to understand the mechanism of pathogenicity of sacbrood virus in the future.

New insights of Sacbrood virus.

Over the last several years, major efforts have been expended to study viral infection of honeybees mainly due to colony losses around the world (Allen M, et al., 1996). It seems that honeybees are infected with numerous viruses mounting to 18 so far. Infection may be asymptomatic but could still have adverse effects on the bee and may even cause death resulting in colony collapse. Sacbrood virus (SBV) is the most widely distributed of all honey bee viruses. Since its first identification in Apis mellifera L in the United State in 1913, infection of SBV has been found in almost all colonies throughout the world (Allen M, et al., 1996; Ellis J D, et al., 2005; Kim Cuc N T, et al., 2008; Ma M X, et al., 2010). SBV infects the brood and adults, but 2-day old larvae appear to be the most susceptible resulting in death (Ritter W, 1996). Diseased larvae do not pupate and ecdysial fl uid collects around the integument in the form of a “sac”, after which the disease has been designated. The color of infected larvae changes from pearly white to pale yellow. Following death, larvae dry out, making a dark brown gondola-shaped scale (Bailey L, 1975). SBV may infect adults without overt signs of disease (Anderson D L, et al., 1989; Bailey L, 1969). However, life span may become shorter (Bailey L, 1969; Wang D I, et al., 1970). Sacbrood occurs most frequently in the spring when the colony is in a very active growing phase (Bailey L, 1969). The SBV was firstly described infecting the eastern honeybee, A. cerana, in Guangdong China in 1972 and was designated the Chinese sacbrood virus (CSBV). It emerged once again in 2008 in Liaoning, China (Ma M X, et al., 2010) causing death to individual bees or the collapse of the whole colony in an epidemic outbreaks. Later, the SBV was identified in A. cerana in Thailand and in Korea in 1982 and 2008, and named Thi SBV (TSBV) and AcSBV-Kor, respectively. SBV is 26-30 nm in diameter, icosahedral, pseudo T=3 structure symmetry, nonenveloped, and featureless in appearance with sedimentation coefficient of 160S and a buoyant density in cesium chloride of 1.33 g, and belongs to Picornaviridae. The outer shell of the capsid is composed of 60 repeated structures, each consisting of one molecule of 3 subunits VP1, VP2, and VP3 (25, 28, and 31.5 kDa), but each consisting of a single molecule of four subunits VP1, VP2, VP3, and VP4 (30.5, 31.5, 37.8 kDa and 44.2 kDa, respectively) about CSBV. The capsid proteins play important roles in the protection and in the determination of host specificity, tissue tropism and regional variations. Based on phylogeny and sequence homology of SBV-VP1, the SBV strains were divided into two major groups, an AC genotype infecting Apis cerana and an AM genotype infecting Apis mellifera, and also that the virus has host specificity and regional variations (Ma M X, et al., 2013).

Preliminary observations on possible pathogen spill-over from Apis mellifera to Apis cerana

Populations of Apis mellifera and Apis cerana in China and Vietnam were surveyed in order to study possible pathogen spill-over from European to Asian honeybees. This is the first survey of the prevalence of honeybee pathogens in apiaries in Vietnam, including pathogen prevalence in wild A. cerana colonies never in contact with A. mellifera. The bee samples were assayed for eight honeybee viruses: deformed wing virus (DWV); black queen cell virus (BQCV); sac brood virus (SBV); acute bee paralysis virus (ABPV); Kashmir bee virus (KBV); Israeli acute paralysis virus (IAPV); chronic bee paralysis virus (CBPV); and slow bee paralysis virus (SBPV), for two gut parasites (Nosema ssp.) and for the causative agent for European foulbrood (Melissococcus plutonius). The Vietnamese samples were assayed for Acarapis woodi infestation. No clear evidence of unique inter-specific transmission of virus infections between the two honeybee species was found. However, in wild A. cerana colonies, the only virus infection detected was DWV. With findings of IAPV infections in Chinese samples of A. cerana colonies in contact with A. mellifera, inter-specific transmission of IAPV cannot be ruled out. BQCV was the most prevalent virus in managed colonies irrespective of bee species. We did not detect the causative agent of European foulbrood, M. plutonius in wild or isolated colonies of A. cerana in Vietnam or China; however, low incidence of this pathogen was found in the Asian host species when in contact with its European sister species. No evidence for the presence of A. woodi was found in the Vietnamese samples.

Replication of Chinese sacbrood virus in primary cell cultures of Asian honeybee (Apis cerana).

A primary cell culture system was established for the first time from embryonic tissues of Asian honeybee, Apis cerana, and used to trace the early infection process of Chinese sacbrood virus (CSBV), an iflavirus in the family Iflaviridae. A monolayer of epithelium-like cells of A. cerana, approximately 8-10 μm in diameter, was grown in Kimura's insect medium at 28 °C within 3-4 days of setting up the cultures. Such cultured cells were inoculated with CSBV purified from infected larvae or pupae for 2 h. In electron and confocal micrographs, viral particles accumulated as filamentous or vesicular inclusions in the cytoplasm of infected cultured cells at 36 h post-inoculation (hpi). Real-time quantitative RT-PCR assay showed that the expression levels of four cistrons of CSBV in the cultured cells increased rapidly between 12 and 48 hpi. This newly established primary cell culture derived from A. cerana will be useful for further studies of infection caused by CSBV.