scholarly journals Dynamic evolution in the key honey bee pathogen Deformed wing virus

2019 ◽  
Author(s):  
Eugene V. Ryabov ◽  
Anna K. Childers ◽  
Dawn Lopez ◽  
Kyle Grubbs ◽  
Francisco Posada-Florez ◽  
...  
Keyword(s):  
Honey Bees ◽  
Selective Sweep ◽  
Competitive Exclusion ◽  
Rna Virus ◽  
Genetic System ◽  
Reverse Genetic System ◽  
Cdna Clones ◽  
Reverse Genetic ◽  
Deformed Wing Virus ◽  
Selection For

AbstractThe impacts of invertebrate RNA virus population dynamics on virulence and infection outcomes are poorly understood. Deformed wing virus (DWV), the main viral pathogen of honey bees, negatively impacts bee health which can lead to colony death. Despite previous reports on the reduction of DWV diversity following the arrival of the parasitic mite Varroa destructor, the key DWV vector, we found high genetic diversity of DWV in infested United States (US) honey bee colonies. Phylogenetic analysis showed that the divergent US DWV genotypes are of monophyletic origin, which were likely generated as a result of diversification after a genetic bottleneck. To investigate the population dynamics of this divergent DWV, we designed a series of novel infectious cDNA clones corresponding to co-existing DWV genotypes, thereby devising a reverse genetic system for an invertebrate RNA virus quasispecies. Equal replication rates were observed for all clone-derived DWV variants in single infections. Surprisingly, individual clones replicated to the same high levels as their mixtures and even the parental highly diverse natural DWV population, suggesting that complementation between genotypes was not required to replicate to high levels. Mixed clone-derived infections showed a lack of strong competitive exclusion, suggesting that the DWV genotypes were adapted to co-exist. Mutational and recombination events were observed across clone progeny providing new insights into the forces that drive and constrain virus diversification. Accordingly, herein we propose a new model of Varroa-induced DWV dynamics whereby an initial selective sweep is followed by virus diversification fueled by negative frequency-dependent selection for new genotypes. This selection likely reflects the ability of rare lineages to evade host defenses, specifically antiviral RNA interference (RNAi). In support of this, we show that RNAi induced against one DWV strain is less effective against an alternate strain from the same population.Author SummaryVirulence of Deformed wing virus (DWV), a major pathogen of honey bees, showed a sharp and significant increase following the introduction of its vector, the mite Varroa destructor. Varroa vectoring resulted in genetic changes of DWV, including reduction of DWV diversity to nearly clonal levels in the UK and Hawaii. Contrary to the previous reports, we discovered that virulent DWV populations circulating across the Varroa-infested United States included many divergent genotypes generated following a strong bottleneck event. We designed a series of the full-length infectious cDNA clones that captured the diversity of a typical virulent DWV population from a declining Varroa-infested colony, effectively establishing first reverse genetic system for an invertebrate RNA virus quasispecies, in order to investigate interactions between the virus genotypes. We demonstrated that individual co-existing DWV genotypes and diverse natural DWV populations replicated equally well indicating that complementation between isolates was not required to enable DWV replication to high levels. Also, no obvious competitive exclusion was detected between genotypes in mixed infections suggesting DWV genotypes are adapted to co-exist to maintain overall population diversity. We suggest that introduction of Varroa resulted in an initial selective sweep of DWV diversity which was followed by DWV diversification driven by selection for new genotypes capable of evading host defenses, specifically antiviral RNA interference.

scholarly journals Development of a Reverse Genetic System to Generate Recombinant Chimeric Tacaribe Virus that Expresses Junín Virus Glycoproteins

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 948
Author(s):  
Sabrina Foscaldi ◽  
María Eugenia Loureiro ◽  
Claudia Sepúlveda ◽  
Carlos Palacios ◽  
María Belén Forlenza ◽  
...  
Keyword(s):  
Hemorrhagic Fever ◽  
Genetic System ◽  
Reverse Genetic System ◽  
Dependent Manner ◽  
Reverse Genetic ◽  
Junin Virus ◽  
Tacaribe Virus ◽  
Growth Properties ◽  
Intracellular Expression ◽  
Junín Virus

Mammarenaviruses are enveloped and segmented negative-stranded RNA viruses that comprise several pathogenic members associated with severe human hemorrhagic fevers. Tacaribe virus (TCRV) is the prototype for the New World group of mammarenaviruses and is not only naturally attenuated but also phylogenetically and antigenically related to all South American pathogenic mammarenaviruses, particularly the Junín virus (JUNV), which is the etiological agent of Argentinian hemorrhagic fever (AHF). Moreover, since TCRV protects guinea pigs and non-human primates from lethal challenges with pathogenic strains of JUNV, it has already been considered as a potential live-attenuated virus vaccine candidate against AHF. Here, we report the development of a reverse genetic system that relies on T7 polymerase-driven intracellular expression of the complementary copy (antigenome) of both viral S and L RNA segments. Using this approach, we successfully recovered recombinant TCRV (rTCRV) that displayed growth properties resembling those of authentic TCRV. We also generated a chimeric recombinant TCRV expressing the JUNV glycoproteins, which propagated similarly to wild-type rTCRV. Moreover, a controlled modification within the S RNA 5′ non-coding terminal sequence diminished rTCRV propagation in a cell-type dependent manner, giving rise to new perspectives where the incorporation of additional attenuation markers could contribute to develop safe rTCRV-based vaccines against pathogenic mammarenaviruses.

scholarly journals Establishment of an efficient reverse genetic system of Mumps virus S79 from cloned DNA

2019 ◽  
Vol 15 (5) ◽  
pp. 499-505 ◽  
Author(s):  
Duo Zhou ◽  
Meng-Ying Zhu ◽  
Yi-Long Wang ◽  
Xiao-Qiang Hao ◽  
Dong-Ming Zhou ◽  
...  
Keyword(s):  
Genetic System ◽  
Mumps Virus ◽  
Reverse Genetic System ◽  
Reverse Genetic

scholarly journals Deformed Wing Virus Infection in Honey Bees (Apis mellifera L.)

2019 ◽  
Vol 56 (4) ◽  
pp. 636-641 ◽  
Author(s):  
Roman V. Koziy ◽  
Sarah C. Wood ◽  
Ivanna V. Kozii ◽  
Claire Janse van Rensburg ◽  
Igor Moshynskyy ◽  
...  
Keyword(s):  
Apis Mellifera ◽  
Honey Bee ◽  
Honey Bees ◽  
Rna Virus ◽  
Mandibular Glands ◽  
Deformed Wing Virus ◽  
Clinically Normal ◽  
Bee Health ◽  
Honey Bee Health ◽  
Honey Bee Workers

Deformed wing virus (DWV) is a single-stranded RNA virus of honey bees ( Apis mellifera L.) transmitted by the parasitic mite Varroa destructor. Although DWV represents a major threat to honey bee health worldwide, the pathological basis of DWV infection is not well documented. The objective of this study was to investigate clinicopathological and histological aspects of natural DWV infection in honey bee workers. Emergence of worker honey bees was observed in 5 colonies that were clinically affected with DWV and the newly emerged bees were collected for histopathology. DWV-affected bees were 2 times slower to emerge and had 30% higher mortality compared to clinically normal bees. Hypopharyngeal glands in bees with DWV were hypoplastic, with fewer intracytoplasmic secretory vesicles; cells affected by apoptosis were observed more frequently. Mandibular glands were hypoplastic and were lined by cuboidal epithelium in severely affected bees compared to tall columnar epithelium in nonaffected bees. The DWV load was on average 1.7 × 106 times higher ( P < .001) in the severely affected workers compared to aged-matched sister honey bee workers that were not affected by deformed wing disease based on gross examination. Thus, DWV infection is associated with prolonged emergence, increased mortality during emergence, and hypoplasia of hypopharyngeal and mandibular glands in newly emerged worker honey bees in addition to previously reported deformed wing abnormalities.

scholarly journals Deformed wing virus type A, a major honey bee pathogen, is vectored by the mite Varroa destructor in a non-propagative manner

2019 ◽  
Author(s):  
Francisco Posada-Florez ◽  
Anna K. Childers ◽  
Matthew C. Heerman ◽  
Noble I. Egekwu ◽  
Steven C. Cook ◽  
...  
Keyword(s):  
Honey Bee ◽  
Honey Bees ◽  
Varroa Destructor ◽  
Rna Virus ◽  
Fold Increase ◽  
Type A ◽  
Deformed Wing Virus ◽  
Negative Strand ◽  
Viral Loads ◽  
Insect Pollinator

AbstractHoney bees, the primary managed insect pollinator, suffer considerable losses due to Deformed wing virus (DWV), an RNA virus vectored by the mite Varroa destructor. Mite vectoring has resulted in the emergence of virulent DWV variants. The basis for such changes in DWV is poorly understood. Most importantly, it remains unclear whether replication of DWV occurs in the mite. In this study, we exposed Varroa mites to DWV type A via feeding on artificially infected honey bees. A significant, 357-fold increase in DWV load was observed in these mites after 2 days. However, after 8 additional days of passage on honey bee pupae with low viral loads, the DWV load dropped by 29-fold. This decrease significantly reduced the mites’ ability to transmit DWV to honey bees. Notably, negative-strand DWV RNA, which could indicate viral replication, was detected only in mites collected from pupae with high DWV levels but not in the passaged mites. We also found that Varroa mites contain honey bee mRNAs, consistent with the acquisition of honey bee cells which would additionally contain DWV replication complexes with negative-strand DWV RNA. We propose that transmission of DWV type A by Varroa mites occurs in a non-propagative manner.

scholarly journals Characterization and reverse genetic establishment of cattle derived Akabane virus in China

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Dongjie Chen ◽  
Di Wang ◽  
Fang Wei ◽  
Yufang Kong ◽  
Junhua Deng ◽  
...  
Keyword(s):  
Genetic System ◽  
Reverse Genetic System ◽  
Akabane Virus ◽  
Reverse Genetic ◽  
Wild Type ◽  
T7 Promoter ◽  
Cytopathic Effects ◽  
The World ◽  
L Segment ◽  
Genome Manipulation

Abstract Background Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. Results An AKAV strain defined as TJ2016 was firstly isolated from the bovine sera in China in 2016. Sequence analysis of the S and M segments suggested that the isolated AKAV strain was closely related to the AKAV strains JaGAr39 and JaLAB39, which belonged to AKAV genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ2016 S, M, and L segment was constructed separately into the TVT7R plasmid at the downsteam of T7 promoter and named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. The above three plasmids were further transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to produce the rescued virus AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to be with similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells. Conclusion We successfully isolated a AKAV strain TJ2016 from the sera of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool for further research on AKAV pathogenesis and even vaccine studies.

scholarly journals Direct Evidence for Infection of Varroa destructor Mites with the Bee-Pathogenic Deformed Wing Virus Variant B - but Not Variant A - via Fluorescence-in situ-Hybridization Analysis.

2020 ◽  
Author(s):  
Sebastian Gisder ◽  
Elke Genersch
Keyword(s):  
Salivary Glands ◽  
Honey Bees ◽  
Varroa Destructor ◽  
Direct Evidence ◽  
Rna Virus ◽  
Colony Losses ◽  
Deformed Wing Virus ◽  
Brain Infection ◽  
Biological Vector

Deformed wing virus (DWV) is a bee pathogenic, single- and positive-stranded RNA virus that has been involved in severe honey bee colony losses worldwide. DWV, when transmitted horizontally or vertically from bee to bee, causes mainly covert infections not associated with any visible symptoms or damage. Overt infections occur after vectorial transmission of DWV to the developing bee pupae through the ectoparasitic mite Varroa destructor. Symptoms of overt infections are pupal death, bees emerging with deformed wings and shortened abdomens, or cognitive impairment due to brain infection. So far, three variants of DWV, DWV-A, DWV-B, and DWV-C, have been described. While it is widely accepted that V. destructor acts as vector of DWV, the question of whether the mite only functions as a mechanical vector or whether DWV can infect the mite thus using it as a biological vector is hotly debated, because in the literature data can be found that support both hypotheses. In order to settle this scientific dispute, we analyzed putatively DWV-infected mites with a newly established protocol for fluorescence-in situ-hybridization of mites and demonstrated DWV-specific signals inside mite cells. We provide compelling and direct evidence that DWV-B infects the intestinal epithelium and the salivary glands of V. destructor. In contrast, no evidence for DWV-A infecting mite cells was found. Our data are key to understanding the pathobiology of DWV, the mite’s role as a biological DWV vector and the quasispecies dynamics of this RNA virus when switching between insect and arachnid host species. IMPORTANCE Deformed wing virus (DWV) is a bee pathogenic, originally rather benign, single- and positive-stranded RNA virus. Only the vectorial transmission of this virus to honey bees by the ectoparasitic mite Varroa destructor leads to fatal or symptomatic infections of individuals, usually followed by collapse of the entire colony. Studies on whether the mite only acts as a mechanical virus vector or whether DWV can infect the mite and thus use it as a biological vector have led to disparate results. In our study using fluorescence-in situ-hybridization we provide compelling and direct evidence that at least the DWV-B variant infects the gut epithelium and the salivary glands of V. destructor. Hence, the host range of DWV includes both, bees (Insecta) and mites (Arachnida). Our data contribute to a better understanding of the triangular relationship between honey bees, V. destructor and DWV and the evolution of virulence in this viral bee pathogen.

scholarly journals Tet-Inducible Production of Infectious Zika Virus from the Full-Length cDNA Clones of African- and Asian-Lineage Strains

Viruses ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 700 ◽  
Author(s):  
Lizhou Zhang ◽  
Wei Ji ◽  
Shuang Lyu ◽  
Luhua Qiao ◽  
Guangxiang Luo
Keyword(s):  
Zika Virus ◽  
Reverse Genetics ◽  
Genetic System ◽  
Full Length ◽  
Reverse Genetic System ◽  
Cdna Clones ◽  
Viral Pathogen ◽  
Stable Plasmid ◽  
Asian Lineage ◽  
Delta Virus

Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged as an important human viral pathogen, causing congenital malformation including microcephaly among infants born to mothers infected with the virus during pregnancy. Phylogenetic analysis suggested that ZIKV can be classified into African and Asian lineages. In this study, we have developed a stable plasmid-based reverse genetic system for robust production of both ZIKV prototype African-lineage MR766 and clinical Asian-lineage FSS13025 strains using a tetracycline (Tet)-controlled gene expression vector. Transcription of the full-length ZIKV RNA is under the control of the Tet-responsive Ptight promoter at the 5′ end and an antigenomic ribozyme of hepatitis delta virus at the 3′ end. The transcription of infectious ZIKV RNA genome was efficiently induced by doxycycline. This novel ZIKV reverse genetics system will be valuable for the study of molecular viral pathogenesis of ZIKV and the development of new vaccines against ZIKV infection.

Construction of a reverse genetic system for porcine astrovirus

2018 ◽  
Vol 163 (6) ◽  
pp. 1511-1518 ◽  
Author(s):  
Yifeng Qin ◽  
Qingli Fang ◽  
Huan Liu ◽  
Chengyuan Ji ◽  
Ying Chen ◽  
...  
Keyword(s):  
Genetic System ◽  
Reverse Genetic System ◽  
Reverse Genetic ◽  
Porcine Astrovirus
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