Development of a multiplex RT-PCR for the simultaneous detection of three viruses of the honeybee (Apis mellifera L.): Acute bee paralysis virus, Black queen cell virus and Sacbrood virus

In recent years, there has been growing evidence that certain types of honeybee viruses could be transmitted between different pollinators. Within a voluntary monitoring programme, 180 honeybee samples (Apis mellifera carnica) were collected from affected apiaries between 2007 and 2018. Also from August 2017 to August 2018, a total 148 samples of healthy bumblebees (Bombus lapidarius, B. pascuorum, B. terrestris, B. lucorum, B. hortorum, B. sylvarum, B. humilis) were collected at four different locations in Slovenia, and all samples were tested by using RT-PCR methods for six honeybee viruses. Direct sequencing of a total 158 positive samples (acute bee paralysis virus (ABPV n = 33), black queen cell virus (BQCV n = 75), sacbrood bee virus (SBV n = 25) and Lake Sinai virus (LSV n = 25)) was performed from obtained RT-PCR products. The genetic comparison of identified positive samples of bumblebees and detected honeybee field strains of ABPV, BQCV, SBV, and LSV demonstrated 98.74% to 100% nucleotide identity between both species. This study not only provides evidence that honeybees and bumblebees are infected with genetically identical or closely related viral strains of four endemically present honeybee viruses but also detected a high diversity of circulating strains in bumblebees, similar as was observed among honeybees. Important new genetic data for endemic strains circulating in honeybees and bumblebees in Slovenia are presented.

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An Investigation of Honey Bee Viruses Prevalence in Managed Honey Bees (Apis mellifera and Apis cerana) Undergone Colony Decline

The Open Microbiology Journal ◽

10.2174/1874285802115010058 ◽

2021 ◽

Vol 15 (1) ◽

pp. 58-66

Author(s):

Chunying Yuan ◽

Xuejian Jiang ◽

Man Liu ◽

Sa Yang ◽

Shuai Deng ◽

...

Keyword(s):

Apis Mellifera ◽

Honey Bee ◽

Clinical Symptoms ◽

Apis Cerana ◽

Rt Pcr ◽

Black Queen Cell Virus ◽

Queen Cell ◽

Bee Virus ◽

Paralysis Virus ◽

Bee Viruses

Objective: In the absence of known clinical symptoms, viruses were considered to be the most probable key pathogens of honey bee. Therefore, the aim of this study was to investigate the prevalence and distribution of honey bee viruses in managed Apis mellifera and Apis cerana in China. Methods: We conducted a screening of 8 honey bee viruses on A. mellifera and A. cerana samples collected from 54 apiaries from 13 provinces in China using RT-PCR. Results: We found that the types and numbers of viral species significantly differed between A. mellifera and A. cerana. Black Queen Cell Virus (BQCV), Chronic Bee Paralysis Virus (CBPV), Apis mellifera filamentous virus (AmFV), and Kakugo virus (DWV-A/KV) were the primary viruses found in A. mellifera colonies, whereas Chinese Sacbrood Bee Virus (CSBV) and Sacbrood Bee Virus (SBV) were the primary viruses found in A. cerana. The percentage infection of BQCV and CSBV were 84.6% and 61.6% in all detected samples. We first detected the occurrences of Varroa destructor virus-1 (VDV-1 or DWV-B) and DWV-A/KV in China but not ABPV in both A. mellifera and A. cerana. Conclusion: This study showed that BQCV and CSBV are the major threat to investigated A. mellifera and A. cerana colonies.

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Detection of Acute Bee Paralysis Virus and Black Queen Cell Virus from Honeybees by Reverse Transcriptase PCR

Applied and Environmental Microbiology ◽

10.1128/aem.67.5.2384-2387.2001 ◽

2001 ◽

Vol 67 (5) ◽

pp. 2384-2387 ◽

Cited By ~ 100

Author(s):

Mongi Benjeddou ◽

Neil Leat ◽

Mike Allsopp ◽

Sean Davison

Keyword(s):

Reverse Transcriptase ◽

Pcr Primers ◽

Rt Pcr ◽

Black Queen Cell Virus ◽

Silica Membrane ◽

Blind Test ◽

Reverse Transcriptase Pcr ◽

Queen Cell ◽

Acute Bee Paralysis Virus ◽

Paralysis Virus

ABSTRACT A reverse transcriptase PCR (RT-PCR) assay was developed for the detection of acute bee paralysis virus (ABPV) and black queen cell virus (BQCV), two honeybee viruses. Complete genome sequences were used to design unique PCR primers within a 1-kb region from the 3′ end of both genomes to amplify a fragment of 900 bp from ABPV and 700 bp from BQCV. The combined guanidinium thiocyanate and silica membrane method was used to extract total RNA from samples of healthy and laboratory-infected bee pupae. In a blind test, RT-PCR successfully identified the samples containing ABPV and BQCV. Sensitivities were approximately 1,600 genome equivalents of purified ABPV and 130 genome equivalents of BQCV.

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The First Detection and Genetic Characterization of Four Different Honeybee Viruses in Wild Bumblebees from Croatia

Pathogens ◽

10.3390/pathogens10070808 ◽

2021 ◽

Vol 10 (7) ◽

pp. 808

Author(s):

Ivana Tlak Gajger ◽

Laura Šimenc ◽

Ivan Toplak

Keyword(s):

Genetic Characterization ◽

Direct Sequencing ◽

Rt Pcr ◽

Black Queen Cell Virus ◽

Deformed Wing Virus ◽

Queen Cell ◽

Acute Bee Paralysis Virus ◽

Virus Strains ◽

Paralysis Virus

To determine the presence and the prevalence of four different honeybee viruses (acute bee paralysis virus—ABPV, black queen cell virus—BQCV, chronic bee paralysis virus—CBPV, deformed wing virus—DWV) in wild bumblebees, pooled randomly selected bumblebee samples were collected from twenty-seven different locations in the territory of Croatia. All samples were prepared and examined using the RT-PCR methods for quantification of mentioned honeybee viruses. Determined prevalence (%) of identified positive viruses were in the following decreasing order: BQCV > DWV > ABPV, CBPV. Additionally, direct sequencing of samples positive for BQCV (n = 24) and DWV (n = 2) was performed, as well as a test of molecular phylogeny comparison with those available in GenBank. Selected positive field viruses’ strains showed 95.7 to 100% (BQCV) and 98.09% (DWV) nucleotide identity with previously detected and deposited honeybee virus strains in the geographic areas in Croatia and neighboring Slovenia. In this article, the first detection of four honeybee viruses with genetic characterization of high diversity strains circulating in wild bumblebees in Croatia is presented.

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The Pathogens Spillover and Incidence Correlation in Bumblebees and Honeybees in Slovenia

Pathogens ◽

10.3390/pathogens10070884 ◽

2021 ◽

Vol 10 (7) ◽

pp. 884

Author(s):

Metka Pislak Ocepek ◽

Ivan Toplak ◽

Urška Zajc ◽

Danilo Bevk

Keyword(s):

Pathogen Transmission ◽

Rt Pcr ◽

Black Queen Cell Virus ◽

Deformed Wing Virus ◽

Nosema Bombi ◽

Crithidia Bombi ◽

Queen Cell ◽

Acute Bee Paralysis Virus ◽

Bee Virus ◽

Paralysis Virus

Slovenia has a long tradition of beekeeping and a high density of honeybee colonies, but less is known about bumblebees and their pathogens. Therefore, a study was conducted to define the incidence and prevalence of pathogens in bumblebees and to determine whether there are links between infections in bumblebees and honeybees. In 2017 and 2018, clinically healthy workers of bumblebees (Bombus spp.) and honeybees (Apis mellifera) were collected on flowers at four different locations in Slovenia. In addition, bumblebee queens were also collected in 2018. Several pathogens were detected in the bumblebee workers using PCR and RT-PCR methods: 8.8% on acute bee paralysis virus (ABPV), 58.5% on black queen cell virus (BQCV), 6.8% on deformed wing virus (DWV), 24.5% on sacbrood bee virus (SBV), 15.6% on Lake Sinai virus (LSV), 16.3% on Nosema bombi, 8.2% on Nosema ceranae, 15.0% on Apicystis bombi and 17.0% on Crithidia bombi. In bumblebee queens, only the presence of BQCV, A. bombi and C. bombi was detected with 73.3, 26.3 and 33.3% positive samples, respectively. This study confirmed that several pathogens are regularly detected in both bumblebees and honeybees. Further studies on the pathogen transmission routes are required.

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Presence and Prevalence of Viruses in Local and Migratory Honeybees (Apis mellifera) in Massachusetts

Applied and Environmental Microbiology ◽

10.1128/aem.01319-09 ◽

2009 ◽

Vol 75 (24) ◽

pp. 7862-7865 ◽

Cited By ~ 29

Author(s):

Anna Welch ◽

Francis Drummond ◽

Sunil Tewari ◽

Anne Averill ◽

John P. Burand

Keyword(s):

Apis Mellifera ◽

Black Queen Cell Virus ◽

Deformed Wing Virus ◽

Sacbrood Virus ◽

Queen Cell ◽

The Impact

ABSTRACT Migratory and local bees in Massachusetts were analyzed for seven viruses. Three were detected: black queen cell virus (BQCV), deformed wing virus (DWV), and sacbrood virus (SBV). DWV was most common, followed closely by BQCV and then by SBV. BQCV and SBV were present at significantly higher rates in the migratory bees assayed, bringing into question the impact that these bees have on the health of local bee populations.

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The Pathogen Profile of a Honey Bee Queen Does Not Reflect That of Her Workers

Insects ◽

10.3390/insects11060382 ◽

2020 ◽

Vol 11 (6) ◽

pp. 382 ◽

Cited By ~ 1

Author(s):

Jessica L. Kevill ◽

Katie Lee ◽

Michael Goblirsch ◽

Erin McDermott ◽

David R. Tarpy ◽

...

Keyword(s):

Honey Bee ◽

Horizontal Transmission ◽

Pathogen Transmission ◽

Black Queen Cell Virus ◽

Deformed Wing Virus ◽

Israeli Acute Paralysis Virus ◽

Queen Cell ◽

Honey Bee Queen ◽

Acute Bee Paralysis Virus ◽

Paralysis Virus

Throughout a honey bee queen’s lifetime, she is tended to by her worker daughters, who feed and groom her. Such interactions provide possible horizontal transmission routes for pathogens from the workers to the queen, and as such a queen’s pathogen profile may be representative of the workers within a colony. To explore this further, we investigated known honey bee pathogen co-occurrence, as well as pathogen transmission from workers to queens. Queens from 42 colonies were removed from their source hives and exchanged into a second, unrelated foster colony. Worker samples were taken from the source colony on the day of queen exchange and the queens were collected 24 days after introduction. All samples were screened for Nosema spp., Trypanosome spp., acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), Israeli acute paralysis virus (IAPV), Lake Sinai virus (LSV), and deformed wing virus master variants (DWV-A, B, and C) using RT-qPCR. The data show that LSV, Nosema, and DWV-B were the most abundant pathogens in colonies. All workers (n = 42) were LSV-positive, 88% were Nosema-positive, whilst pathogen loads were low (<1 × 106 genome equivalents per pooled worker sample). All queens (n = 39) were negative for both LSV and Nosema. We found no evidence of DWV transmission occurring from worker to queen when comparing queens to foster colonies, despite DWV being present in both queens and workers. Honey bee pathogen presence and diversity in queens cannot be revealed from screening workers, nor were pathogens successfully transmitted to the queen.

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

The Canadian Entomologist ◽

10.4039/tce.2015.23 ◽

2015 ◽

Vol 148 (1) ◽

pp. 22-35 ◽

Cited By ~ 4

Author(s):

Suresh D. Desai ◽

Santosh Kumar ◽

Robert W. Currie

Keyword(s):

Polymerase Chain Reaction ◽

Honey Bee ◽

Multiple Infections ◽

Black Queen Cell Virus ◽

Chain Reaction ◽

Deformed Wing Virus ◽

Sacbrood Virus ◽

Queen Cell ◽

Polymerase Chain ◽

Paralysis Virus

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.

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