scholarly journals DWV Infection in vitro Using Honey Bee Pupal Tissue

2021 ◽  
Vol 12 ◽  
Author(s):  
Yunfei Wu ◽  
Xuye Yuan ◽  
Jing Li ◽  
Tatsuhiko Kadowaki
Keyword(s):  
Viral Infection ◽  
Honey Bee ◽  
Host Cells ◽  
Rna Dependent Rna Polymerase ◽  
Deformed Wing Virus ◽  
Polyprotein Processing ◽  
3C Protease ◽  
Undifferentiated Cells ◽  
Bee Viruses

The deformed wing virus (DWV) has been best characterized among honey bee viruses; however, very little is known regarding the mechanisms of viral infection and replication due to the lack of immortalized honey bee cell lines. To solve this problem, we established an in vitro system using honey bee pupal tissue to reconstruct DWV binding and entry into the host cell, followed by translation of the RNA genome and polyprotein processing using RNA-dependent RNA polymerase (RdRP) as a marker. Using this system, the P-domain of the virion subunit VP1 was found to be essential for DWV infection, but not for binding and entry into the cell. DWV efficiently infected the head tissue derived from early but not late pupa, suggesting that undifferentiated cells are targeted for viral infection. Furthermore, we found that inhibitors of mammalian picornavirus 3C-protease, rupintrivir and quercetin suppressed RdRP synthesis, indicating that this in vitro system is also useful for screening a compound to control viral infection. Our in vitro system may help to understand the mechanism of DWV infection in host cells.

scholarly journals DWV infection and replication at the early stage in vitro using honey bee pupal cells

2020 ◽  
Author(s):  
Yunfei Wu ◽  
Jing Li ◽  
Tatsuhiko Kadowaki
Keyword(s):  
Viral Infection ◽  
Honey Bee ◽  
Large Scale ◽  
Early Stage ◽  
Deformed Wing Virus ◽  
System P ◽  
Undifferentiated Cells ◽  
Prevalent Virus ◽  
Bee Colonies

AbstractDeformed wing virus (DWV) has been best characterized among honey bee viruses; however, very little is known about the mechanisms of viral infection and replication due to the lack of honey bee cell lines. To resolve this problem, we established in vitro system to reconstitute DWV binding and entry to the host cell followed by translation of the genome RNA and the polyprotein processing with honey bee pupal cells. Using this system, P-domain of VP1 was found to be essential for DWV infection/replication but not binding/entry to the cell. DWV efficiently infects/replicates in cells derived from early but not late pupa, suggesting that the undifferentiated cells are targeted for the viral infection/replication. Furthermore, we found that inhibitors for mammalian picornavirus 3C-Protease, Rupintrivir and Quercetin suppress DWV infection/replication, indicating that this in vitro system is also useful for screening a compound to modify the viral infection/replication. Our in vitro system should help to understand the mechanisms of DWV infection and replication at the early stage.ImportanceRecent decline of managed honey bee colonies has been driven by the pathogens and parasites. However, studying the mechanisms of pathogen infection and replication in honey bee at molecular and cellular levels has been challenging. DWV is the most prevalent virus in honey bee across the globe and we established in vitro system to reconstitute the viral infection and replication with the primary pupal cells. Using RNA-dependent RNA polymerase (RdRP) and the negative strand of DWV genome RNA as markers, we show that the pupal cells can support DWV infection and at least replication at the early stage. The results shown in this report indicate that our in vitro system helps to uncover the mechanisms of DWV infection and replication. Furthermore, it is also feasible to conduct a large scale screening for compounds to inhibit or stimulate DWV infection/replication.

scholarly journals A novel system for maintaining Varroa destructor mites on artificial diets and its application for studying mites as a vector for honey bee viruses

2019 ◽  
Author(s):  
Francisco Posada-Florez ◽  
Eugene V. Ryabov ◽  
Matthew C. Heerman ◽  
Yanping Chen ◽  
Jay D. Evans ◽  
...  
Keyword(s):  
Honey Bee ◽  
Varroa Destructor ◽  
Artificial Diet ◽  
Artificial Diets ◽  
Deformed Wing Virus ◽  
Limited Success ◽  
Honey Bee Health ◽  
Serious Injury ◽  
Bee Viruses

AbstractThe mite Varroa destructor is one of the most destructive parasites of the honey bee (Apis mellifera) and the primary cause of colony collapse in most regions of the world. These mites cause serious injury to their hosts, especially during the larval and pupal stages, and serve as the vector for several viruses, which affect honey bee health causing colony death. Attempts by beekeepers to control these mites have yielded limited success. The inability to rear populations of mites in vitro that excludes contact with their honey bee hosts has stymied research of Varroa biology. Previous attempts to rear and/or maintain Varroa mites in vitro by feeding them on artificial diets have had limited success. Several methods were plagued by mechanical failures including leaking membranes and, thus far, none have been widely adopted. Here we report a robust system for maintaining Varroa mites that includes an artificial diet, which does not contain honey bee tissue-derived components, thus making it particularly valuable in studying mite vectoring of honey bee viruses. With our system we demonstrated for the first time that Varroa mites maintained on an artificial diet supplemented with the particles of honey bee viruses, cDNA clone-derived genetically tagged Varroa destructor virus-1 and wild-type Deformed wing virus, can acquire and later transmit these viruses to recipient honey bee pupae. Along with providing an opportunity to study parasites and pathogens in the absence of honey bee hosts, this in vitro system for Varroa mite maintenance is both scalable and consistent. These features can be used to better understand mite nutritional needs, metabolic activity, responses to chemicals and other biological functions.

scholarly journals Varroa destructor mites vector and transmit pathogenic honey bee viruses acquired from an artificial diet

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242688
Author(s):  
Francisco Posada-Florez ◽  
Eugene V. Ryabov ◽  
Matthew C. Heerman ◽  
Yanping Chen ◽  
Jay D. Evans ◽  
...  
Keyword(s):  
Honey Bee ◽  
Varroa Destructor ◽  
Artificial Diet ◽  
Fat Body ◽  
Deformed Wing Virus ◽  
Virus Particles ◽  
Colony Collapse ◽  
Cellular Components ◽  
Bee Viruses

The ectoparasitic mite Varroa destructor is one of the most destructive pests of the honey bee (Apis mellifera) and the primary biotic cause of colony collapse in many regions of the world. These mites inflict physical injury on their honey bee hosts from feeding on host hemolymph and fat body cells/cellular components, and serve as the vector for deadly honey bee viruses, including Deformed wing virus (DWV) and the related Varroa destructor virus-1 (VDV-1) (i.e., DWV-like viruses). Studies focused on elucidating the dynamics of Varroa-mediated vectoring and transmission of DWV-like viruses may be confounded by viruses present in ingested host tissues or the mites themselves. Here we describe a system that includes an artificial diet free of insect tissue-derived components for maintaining Varroa mites for in vitro experimentation. Using this system, together with the novel engineered cDNA clone-derived genetically tagged VDV-1 and wild-type DWV, we demonstrated for the first time that Varroa mites provided an artificial diet supplemented with engineered viruses for 36 hours could acquire and transmit sufficient numbers of virus particles to establish an infection in virus-naïve hosts. While the in vitro system described herein provides for only up to five days of mite survival, precluding study of the long-term impacts of viruses on mite health, the system allows for extensive insights into the dynamics of Varroa-mediated vectoring and transmission of honey bee viruses.

The Use of Lectins as Tools to Combat SARS-CoV-2

2021 ◽  
Vol 27 ◽  
Author(s):  
Daniela Martinez ◽  
Diego Amaral ◽  
David Markovitz ◽  
Luciano Pinto
Keyword(s):  
Viral Infection ◽  
Host Cells ◽  
Viral Fusion ◽  
Cell Receptors ◽  
First Case ◽  
Mannose Binding ◽  
New Treatments ◽  
Viral Surface

Background: in december 2019, china announced the first case of an infection caused by an, until then, unknown virus: sars-cov-2. since then, researchers have been looking for viable alternatives for the treatment and/or cure of viral infection. among the possible complementary solutions are lectins, and proteins that are reversibly bound to different carbohydrates. the spike protein, present on the viral surface, can interact with different cell receptors: ace2, cd147, and dc-signr. since lectins have an affinity for different carbohydrates, the binding with the glycosylated cell receptors represents a possibility of preventing the virus from binding to the receptors of host cells. Objective: in this review we discuss the main lectins that are possible candidates for use in the treatment of covid-19, highlighting those that have already demonstrated antiviral activity in vivo and in vitro, including mannose-binding lectin, griffithsin, banlec, and others. we also aim to discuss the possible mechanism of action of lectins, which appears to occur through the mediation of viral fusion in host cells, by binding of lectins to glycosylated receptors found in human cells and/or binding of these proteins with the spike glycoprotein, present in virus surface.moreover, we also discuss the use of lectins in clinical practice. Conclusion: Even with the development of effective vaccines, new cases of viral infection with the same virus, or new outbreaks with different viruses can occur; so, the development of new treatments should not be discarded. moreover, the discussions made in this work are relevant regarding the anti-viral properties of lectins.

scholarly journals Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp12/7/8 RNA-dependent RNA Polymerase

2021 ◽  
Author(s):  
Agustina P. Bertolin ◽  
Florian Weissmann ◽  
Jingkun Zeng ◽  
Viktor Posse ◽  
Jennifer C. Milligan ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Virus ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Etiologic Agent ◽  
Host Cells ◽  
Rdrp Activity ◽  
Chemical Library ◽  
Rna Dependent Rna Polymerase

SummaryThe coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologs in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer (FRET)-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified 3 novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.

scholarly journals A survey of deformed wing virus and acute bee paralysis virus in honey bee colonies from serbia using real-time RT-PCR

2014 ◽  
Vol 64 (1) ◽  
pp. 81-92 ◽  
Author(s):  
Predrag Simeunović ◽  
Jevrosima Stevanović ◽  
Dejan Vidanović ◽  
Jakov Nišavić ◽  
Dejan Radović ◽  
...  
Keyword(s):  
Real Time ◽  
Honey Bee ◽  
Sampling Location ◽  
Rt Pcr ◽  
Deformed Wing Virus ◽  
Bee Colonies ◽  
Acute Bee Paralysis Virus ◽  
Paralysis Virus ◽  
Bee Viruses

Abstract In this study 55 honey bee colonies from different Serbian regions were monitored for the presence of Deformed Wing Virus (DWV) and Acute Bee Paralysis Virus (ABPV) using TaqMan-based real-time RT-PCR assay. The results revealed the presence of DWV in each sampling location, and ABPV in 10 out of 11 apiaries. High frequency of DWV (76.4%) and ABPV (61.8%) positive samples in asymptomatic colonies can be the consequence of inefficient and postponed Varroa treatment concerning the role of this mite in the transmission and activation of honey bee viruses. The real-time RTPCR technique described in this paper is proved to be the most reliable method for this kind of investigation.

scholarly journals Deformed Wing Virus in Two Widespread Invasive Ants: Geographical Distribution, Prevalence, and Phylogeny

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1309
Author(s):  
Chun-Yi Lin ◽  
Chih-Chi Lee ◽  
Yu-Shin Nai ◽  
Hung-Wei Hsu ◽  
Chow-Yang Lee ◽  
...  
Keyword(s):  
Geographical Distribution ◽  
Honey Bee ◽  
Distribution Patterns ◽  
Transmission Efficiency ◽  
Reservoir Host ◽  
Asia Pacific ◽  
Invasive Ants ◽  
Pollination Services ◽  
Deformed Wing Virus ◽  
Bee Viruses

Spillover of honey bee viruses have posed a significant threat to pollination services, triggering substantial effort in determining the host range of the viruses as an attempt to understand the transmission dynamics. Previous studies have reported infection of honey bee viruses in ants, raising the concern of ants serving as a reservoir host. Most of these studies, however, are restricted to a single, local ant population. We assessed the status (geographical distribution/prevalence/viral replication) and phylogenetic relationships of honey bee viruses in ants across the Asia–Pacific region, using deformed wing virus (DWV) and two widespread invasive ants, Paratrechina longicornis and Anoplolepis gracilipes, as the study system. DWV was detected in both ant species, with differential geographical distribution patterns and prevenance levels between them. These metrics, however, are consistent across the geographical range of the same ant species. Active replication was only evident in P. longicornis. We also showed that ant-associated DWV is genetically similar to that isolated from Asian populations of honey bees, suggesting that local acquisition of DWV by the invasive ants may have been common at least in some of our sampled regions. Transmission efficiency of DWV to local arthropods mediated by ant, however, may vary across ant species.

scholarly journals Molecular detection and phylogenetic assessment of six honeybee viruses in Apis mellifera L. colonies in Bulgaria

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5077 ◽  
Author(s):  
Rositsa Shumkova ◽  
Boyko Neov ◽  
Daniela Sirakova ◽  
Ani Georgieva ◽  
Dimitar Gadjev ◽  
...  
Keyword(s):  
Honey Bee ◽  
Severe Infection ◽  
Eastern Region ◽  
Sequencing Data ◽  
Deformed Wing Virus ◽  
Queen Cell ◽  
Kashmir Bee Virus ◽  
Bee Colonies ◽  
Paralysis Virus ◽  
Bee Viruses

Honey bee colonies suffer from various pathogens, including honey bee viruses. About 24 viruses have been reported so far. However, six of them are considered to cause severe infection which inflicts heavy losses on beekeeping. The aim of this study was to investigate incidence of six honey bee viruses: deformed wing virus (DWV), acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV), sacbrood virus (SBV), kashmir bee virus (KBV), and black queen cell virus (BQCV) by a reverse transcription polymerase chain reaction (RT-PCR). A total of 250 adult honey bee samples were obtained from 50 colonies from eight apiaries situated in three different parts of the country (South, North and West Bulgaria). The results showed the highest prevalence of DWV followed by SBV and ABPV, and one case of BQCV. A comparison with homology sequences available in GenBank was performed by phylogenetic analysis, and phylogenetic relationships were discussed in the context of newly described genotypes in the uninvestigated South Eastern region of Europe. In conclusion, the present study has been the first to provide sequencing data and phylogenetics analyses of some honey bee viruses in Bulgaria.

scholarly journals DWV 3C protease uncovers the diverse catalytic triad in insect RNA viruses

2022 ◽  
Author(s):  
Xuye Yuan ◽  
Tatsuhiko Kadowaki
Keyword(s):  
Honey Bees ◽  
Rna Viruses ◽  
Catalytic Triad ◽  
Enzymatic Properties ◽  
Host Cells ◽  
Deformed Wing Virus ◽  
Mutant Proteins ◽  
3C Protease ◽  
Infected Cells ◽  
And Function

Deformed wing virus (DWV) is the most prevalent Iflavirus that is infecting honey bees worldwide. However, the mechanisms of its infection and replication in host cells are poorly understood. In this study, we analyzed the structure and function of DWV 3C protease (3Cpro), which is necessary for the cleavage of the polyprotein to synthesize mature viral proteins. We found that the 3Cpros of DWV and picornaviruses share common enzymatic properties, including sensitivity to the same inhibitors, such as rupintrivir. The predicted structure of DWV 3Cpro by AlphaFold2, the predicted rupintrivir binding domain, and the protease activities of mutant proteins revealed that it has a Cys-His-Asn catalytic triad. Moreover, 3Cpros of other Iflaviruses and Dicistrovirus appear to contain Asn, Ser, Asp, or Glu as the third residue of the catalytic triad, suggesting diversity in insect RNA viruses. Both precursor 3Cpro with RNA-dependent RNA polymerase and mature 3Cpro are present in DWV-infected cells, suggesting that they may have different enzymatic properties and functions. DWV 3Cpro is the first 3Cpro characterized among insect RNA viruses, and our study uncovered both the common and unique characteristics among 3Cpros of Picornavirales. Furthermore, the specific inhibitors of DWV 3Cpro could be used to control DWV infection in honey bees.

scholarly journals Study of Morphological Features in Pre-Imaginal Honey Bee Impaired by Varroa destructor by Means of Computer Tomography

Insects ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 717
Author(s):  
Tamás Sipos ◽  
Tamás Donkó ◽  
Ildikó Jócsák ◽  
Sándor Keszthelyi
Keyword(s):  
Viral Infection ◽  
Honey Bee ◽  
Varroa Destructor ◽  
Developmental Disorders ◽  
Essential Element ◽  
Total Body Length ◽  
Pcr Analysis ◽  
Brood Cell ◽  
Deformed Wing Virus ◽  
Total Body

The honey bee (Apis mellifera L. 1778) is an essential element in maintaining the diversity of the biosphere and food production. One of its most important parasites is Varroa destructor, Anderson and Trueman, 2000, which plays a role in the vectoring of deformed wing virus (DWV) in honey bee colonies. Our aim was to measure the potential morphometric changes in the pre-imaginal stage of A. mellifera caused by varroosis by means of computed tomography, hence supplying evidence for the presumable role that V. destructor plays as a virus vector. Based on our results, the developmental disorders in honey bees that ensued during the pre-imaginal stages were evident. The total-body length and abdomen length of parasitized specimens were shorter than those of their intact companions. In addition, the calculated quotients of the total-body/abdomen, head/thorax, and head/abdomen in parasitized samples were significantly altered upon infestation. In our view, these phenotypical disorders can also be traced to viral infection mediated by parasitism, which was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Capitalizing on a non-destructive method, our study reveals the deformation of the honey bee due to mite parasitism and the intermediary role this pest plays in viral infection, inside the brood cell.

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