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

Several RNA viruses, including SARS-CoV-2, can infect or use the eye as an entry portal to cause ocular or systemic diseases. Povidone-Iodine (PVP-I) is routinely used during ocular surgeries and eye banking as a cost-effective disinfectant due to its broad-spectrum antimicrobial activity, including against viruses. However, whether PVP-I can exert antiviral activities in virus-infected cells remains elusive. In this study, using Zika (ZIKV) and Chikungunya (CHIKV) virus infection of human corneal and retinal pigment epithelial cells, we report antiviral mechanisms of PVP-I. Our data showed that PVP-I, even at the lowest concentration (0.01%), drastically reduced viral replication in corneal and retinal cells without causing cellular toxicity. Antiviral effects of PVP-I against ZIKV and CHIKV were mediated by direct viral inactivation, thus attenuating the ability of the virus to infect host cells. Moreover, one-minute PVP-I exposure of infected ocular cells drastically reduced viral replication and the production of infectious progeny virions. Furthermore, viral-induced (CHIKV) expression of inflammatory genes (TNF-α, IL-6, IL-8, and IL1β) were markedly reduced in PVP-I treated corneal epithelial cells. Together, our results demonstrate potent antiviral effects of PVP-I against ZIKV and CHIKV infection of ocular cells. Thus, a low dose of PVP-I can be used during tissue harvesting for corneal transplants to prevent potential transmission of RNA viruses via infected cells.

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Multi-host viruses in Argentine ants and honey bees: Increased viral disease in honey bees is associated with Argentine ants

10.26686/wgtn.16912519 ◽

2021 ◽

Author(s):

◽

Jana Dobelmann

Keyword(s):

Honey Bees ◽

Rna Viruses ◽

Population Control ◽

Argentine Ant ◽

Viral Dynamics ◽

Invasive Ants ◽

Argentine Ants ◽

Deformed Wing Virus ◽

Tomato Ringspot Virus ◽

Bee Health

Emerging infectious diseases threaten public health, livestock economies, and wildlife. Human-mediated species introductions can alter host and pathogen communities that shape the dynamics of infectious diseases. Several RNA viruses that have been linked to population declines in wild pollinators and losses of managed honey bees have been detected in multiple other species and are suspected to circulate within insect communities. Yet, we lack an understanding of how disease dynamics are affected by the introduction of novel species. These introduced species include invasive ants, which can disturb honey bees and become a pest in apiaries. The Argentine ant (Linepithema humile) is a globally successful invader that has been observed to attack bees and multiple bee-associated viruses have been detected in this ant species. Here, I studied interactions between Argentine ants and European honey bees (Apis mellifera) and how these interactions affect viral dynamics in beehives. I first tested a range of pollinators and associated insects for RNA viruses that are pathogenic to honey bees. Bee-associated viruses showed evidence for active viral replication in several pollinator species but also in species that cohabit in beehives such as ants, spiders, and cockroaches. Using phylogenetic analyses, I found that viral transmission within communities was shaped by geographic origin rather than being restricted by species barriers. Next, I used a longitudinal field study to test whether Argentine ant presence affected pathogen infections and survival in beehives. Argentine ants tested positive for three bee-associated viruses even before beehives were moved into ant-infested sites. Increased levels of deformed wing virus in beehives in autumn were associated with ant presence, although hive mortality was not affected by ants over the duration of this experiment. I used RNA sequencing on a subset of honey bee samples collected during autumn to study the RNA virome and identify transcriptomic responses associated with ant presence. Twelve RNA viruses were found in beehives, among those, three plant-associated viruses and an unclassified RNA virus that had not previously been observed in honey bees. Deformed wing virus showed the highest viral titres in most hives, but was only marginally affected by ant presence. Sacbrood virus and tomato ringspot virus levels were increased in hives with ants, however, both viruses are not known to infect Argentine ants and the plant-associated tomato ringspot virus seems unlikely to affect bee health. Lastly, I tested the feasibility of controlling Argentine ants in apiaries using a novel pest control strategy. RNA interference is a conserved cellular gene regulation mechanism that could be used to silence specific genes in ants. Using double-stranded RNA (dsRNA) to silence two immune-related genes in Argentine ants was expected to increase pathogen susceptibility, which could then lead to higher pathogen levels that reduce ant numbers. My results indicated that no consistent immune silencing could be achieved in the field. Immune gene expression changes were observed, but pathogen titres were not affected, and ant numbers stayed high. Argentine ant control using a conventional insecticide significantly increased bee survival, whereas many hives in the dsRNA and control group abandoned their hives due to ant attacks. Although population control was not successful using the two Argentine ant-specific dsRNAs, insights into ant immunity and ant-bee interactions could improve the development of novel control strategies. Bee-associated viruses have repeatedly been detected in ant species, yet, this is one of the first studies to investigate whether ants affect viral dynamics in honey bees. I showed that invasive Argentine ants are associated with increases in viral pathogens in honey bees. The mechanisms by which ants affect bee disease are unknown, although there is some evidence for ants transmitting viruses or causing stress responses in bees that affect immunity. The findings of this thesis highlight the risk of invasive ant species disrupting pollination services. New and environmentally-friendly methods to control invasive species are urgently needed to improve bee health and limit the spread of invasive ants, such as Argentine ants. The high prevalence of bee-associated viruses and viral diversity in ants suggests that pathogens that are suitable for population control might be present in ant populations, although risks of spillovers into other species need to be carefully considered.

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A Diverse Range of Novel RNA Viruses in Geographically Distinct Honey Bee Populations

Journal of Virology ◽

10.1128/jvi.00158-17 ◽

2017 ◽

Vol 91 (16) ◽

Cited By ~ 58

Author(s):

Emily J. Remnant ◽

Mang Shi ◽

Gabriele Buchmann ◽

Tjeerd Blacquière ◽

Edward C. Holmes ◽

...

Keyword(s):

Disease Management ◽

Honey Bee ◽

Small Rna ◽

Honey Bees ◽

Rna Viruses ◽

Viral Diversity ◽

Colony Losses ◽

Diverse Range ◽

Deformed Wing Virus ◽

Content Type

ABSTRACT Understanding the diversity and consequences of viruses present in honey bees is critical for maintaining pollinator health and managing the spread of disease. The viral landscape of honey bees (Apis mellifera) has changed dramatically since the emergence of the parasitic mite Varroa destructor, which increased the spread of virulent variants of viruses such as deformed wing virus. Previous genomic studies have focused on colonies suffering from infections by Varroa and virulent viruses, which could mask other viral species present in honey bees, resulting in a distorted view of viral diversity. To capture the viral diversity within colonies that are exposed to mites but do not suffer the ultimate consequences of the infestation, we examined populations of honey bees that have evolved naturally or have been selected for resistance to Varroa. This analysis revealed seven novel viruses isolated from honey bees sampled globally, including the first identification of negative-sense RNA viruses in honey bees. Notably, two rhabdoviruses were present in three geographically diverse locations and were also present in Varroa mites parasitizing the bees. To characterize the antiviral response, we performed deep sequencing of small RNA populations in honey bees and mites. This provided evidence of a Dicer-mediated immune response in honey bees, while the viral small RNA profile in Varroa mites was novel and distinct from the response observed in bees. Overall, we show that viral diversity in honey bee colonies is greater than previously thought, which encourages additional studies of the bee virome on a global scale and which may ultimately improve disease management. IMPORTANCE Honey bee populations have become increasingly susceptible to colony losses due to pathogenic viruses spread by parasitic Varroa mites. To date, 24 viruses have been described in honey bees, with most belonging to the order Picornavirales. Collapsing Varroa-infected colonies are often overwhelmed with high levels of picornaviruses. To examine the underlying viral diversity in honey bees, we employed viral metatranscriptomics analyses on three geographically diverse Varroa-resistant populations from Europe, Africa, and the Pacific. We describe seven novel viruses from a range of diverse viral families, including two viruses that are present in all three locations. In honey bees, small RNA sequences indicate that these viruses are processed by Dicer and the RNA interference pathway, whereas Varroa mites produce strikingly novel small RNA patterns. This work increases the number and diversity of known honey bee viruses and will ultimately contribute to improved disease management in our most important agricultural pollinator.

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Formation and Function of Liquid-Like Viral Factories in Negative-Sense Single-Stranded RNA Virus Infections

Viruses ◽

10.3390/v13010126 ◽

2021 ◽

Vol 13 (1) ◽

pp. 126

Author(s):

Justin M. Su ◽

Maxwell Z. Wilson ◽

Charles E. Samuel ◽

Dzwokai Ma

Keyword(s):

Rna Viruses ◽

Rna Virus ◽

Host Responses ◽

Future Research ◽

Virus Infections ◽

Infected Cells ◽

And Function ◽

Negative Sense ◽

Liquid Liquid Phase Separation

Liquid–liquid phase separation (LLPS) represents a major physiochemical principle to organize intracellular membrane-less structures. Studies with non-segmented negative-sense (NNS) RNA viruses have uncovered a key role of LLPS in the formation of viral inclusion bodies (IBs), sites of viral protein concentration in the cytoplasm of infected cells. These studies further reveal the structural and functional complexity of viral IB factories and provide a foundation for their future research. Herein, we review the literature leading to the discovery of LLPS-driven formation of IBs in NNS RNA virus-infected cells and the identification of viral scaffold components involved, and then outline important questions and challenges for IB assembly and disassembly. We discuss the functional implications of LLPS in the life cycle of NNS RNA viruses and host responses to infection. Finally, we speculate on the potential mechanisms underlying IB maturation, a phenomenon relevant to many human diseases.

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Transcriptomic Responses of the Honey Bee Brain to Infection with Deformed Wing Virus

Viruses ◽

10.3390/v13020287 ◽

2021 ◽

Vol 13 (2) ◽

pp. 287

Author(s):

Marie C. Pizzorno ◽

Kenneth Field ◽

Amanda L. Kobokovich ◽

Phillip L. Martin ◽

Riju A. Gupta ◽

...

Keyword(s):

Apis Mellifera ◽

Honey Bees ◽

Behavioral Changes ◽

Rna Seq ◽

Transcriptomic Response ◽

Viral Pathogen ◽

Deformed Wing Virus ◽

And Function ◽

G Protein Coupled ◽

The Brain

Managed colonies of European honey bees (Apis mellifera) are under threat from Varroa destructor mite infestation and infection with viruses vectored by mites. In particular, deformed wing virus (DWV) is a common viral pathogen infecting honey bees worldwide that has been shown to induce behavioral changes including precocious foraging and reduced associative learning. We investigated how DWV infection of bees affects the transcriptomic response of the brain. The transcriptomes of individual brains were analyzed using RNA-Seq after experimental infection of newly emerged adult bees with DWV. Two analytical methods were used to identify differentially expressed genes from the ~15,000 genes in the Apis mellifera genome. The 269 genes that had increased expression in DWV infected brains included genes involved in innate immunity such as antimicrobial peptides (AMPs), Ago2, and Dicer. Single bee brain NMR metabolomics methodology was developed for this work and indicates that proline is strongly elevated in DWV infected brains, consistent with the increased presence of the AMPs abaecin and apidaecin. The 1361 genes with reduced expression levels includes genes involved in cellular communication including G-protein coupled, tyrosine kinase, and ion-channel regulated signaling pathways. The number and function of the downregulated genes suggest that DWV has a major impact on neuron signaling that could explain DWV related behavioral changes.

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DWV Infection in vitro Using Honey Bee Pupal Tissue

Frontiers in Microbiology ◽

10.3389/fmicb.2021.631889 ◽

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.

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Multi-host viruses in Argentine ants and honey bees: Increased viral disease in honey bees is associated with Argentine ants

10.26686/wgtn.16912519.v1 ◽

2021 ◽

Author(s):

◽

Jana Dobelmann

Keyword(s):

Honey Bees ◽

Rna Viruses ◽

Population Control ◽

Argentine Ant ◽

Viral Dynamics ◽

Invasive Ants ◽

Argentine Ants ◽

Deformed Wing Virus ◽

Tomato Ringspot Virus ◽

Bee Health

Emerging infectious diseases threaten public health, livestock economies, and wildlife. Human-mediated species introductions can alter host and pathogen communities that shape the dynamics of infectious diseases. Several RNA viruses that have been linked to population declines in wild pollinators and losses of managed honey bees have been detected in multiple other species and are suspected to circulate within insect communities. Yet, we lack an understanding of how disease dynamics are affected by the introduction of novel species. These introduced species include invasive ants, which can disturb honey bees and become a pest in apiaries. The Argentine ant (Linepithema humile) is a globally successful invader that has been observed to attack bees and multiple bee-associated viruses have been detected in this ant species. Here, I studied interactions between Argentine ants and European honey bees (Apis mellifera) and how these interactions affect viral dynamics in beehives. I first tested a range of pollinators and associated insects for RNA viruses that are pathogenic to honey bees. Bee-associated viruses showed evidence for active viral replication in several pollinator species but also in species that cohabit in beehives such as ants, spiders, and cockroaches. Using phylogenetic analyses, I found that viral transmission within communities was shaped by geographic origin rather than being restricted by species barriers. Next, I used a longitudinal field study to test whether Argentine ant presence affected pathogen infections and survival in beehives. Argentine ants tested positive for three bee-associated viruses even before beehives were moved into ant-infested sites. Increased levels of deformed wing virus in beehives in autumn were associated with ant presence, although hive mortality was not affected by ants over the duration of this experiment. I used RNA sequencing on a subset of honey bee samples collected during autumn to study the RNA virome and identify transcriptomic responses associated with ant presence. Twelve RNA viruses were found in beehives, among those, three plant-associated viruses and an unclassified RNA virus that had not previously been observed in honey bees. Deformed wing virus showed the highest viral titres in most hives, but was only marginally affected by ant presence. Sacbrood virus and tomato ringspot virus levels were increased in hives with ants, however, both viruses are not known to infect Argentine ants and the plant-associated tomato ringspot virus seems unlikely to affect bee health. Lastly, I tested the feasibility of controlling Argentine ants in apiaries using a novel pest control strategy. RNA interference is a conserved cellular gene regulation mechanism that could be used to silence specific genes in ants. Using double-stranded RNA (dsRNA) to silence two immune-related genes in Argentine ants was expected to increase pathogen susceptibility, which could then lead to higher pathogen levels that reduce ant numbers. My results indicated that no consistent immune silencing could be achieved in the field. Immune gene expression changes were observed, but pathogen titres were not affected, and ant numbers stayed high. Argentine ant control using a conventional insecticide significantly increased bee survival, whereas many hives in the dsRNA and control group abandoned their hives due to ant attacks. Although population control was not successful using the two Argentine ant-specific dsRNAs, insights into ant immunity and ant-bee interactions could improve the development of novel control strategies. Bee-associated viruses have repeatedly been detected in ant species, yet, this is one of the first studies to investigate whether ants affect viral dynamics in honey bees. I showed that invasive Argentine ants are associated with increases in viral pathogens in honey bees. The mechanisms by which ants affect bee disease are unknown, although there is some evidence for ants transmitting viruses or causing stress responses in bees that affect immunity. The findings of this thesis highlight the risk of invasive ant species disrupting pollination services. New and environmentally-friendly methods to control invasive species are urgently needed to improve bee health and limit the spread of invasive ants, such as Argentine ants. The high prevalence of bee-associated viruses and viral diversity in ants suggests that pathogens that are suitable for population control might be present in ant populations, although risks of spillovers into other species need to be carefully considered.

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Subgenomic Negative-Strand RNA Function during Mouse Hepatitis Virus Infection

Journal of Virology ◽

10.1128/jvi.74.9.4039-4046.2000 ◽

2000 ◽

Vol 74 (9) ◽

pp. 4039-4046 ◽

Cited By ~ 58

Author(s):

Ralph S. Baric ◽

Boyd Yount

Keyword(s):

Hepatitis Virus ◽

Mouse Hepatitis Virus ◽

Full Length ◽

Host Cells ◽

Positive Strand ◽

Negative Strand ◽

Molar Ratios ◽

Infected Cells ◽

And Function ◽

Hepatitis Virus Infection

ABSTRACT Mouse hepatitis virus (MHV)-infected cells contain full-length and subgenomic-length positive- and negative-strand RNAs. The origin and function of the subgenomic negative-strand RNAs is controversial. In this report we demonstrate that the synthesis and molar ratios of subgenomic negative strands are similar in alternative host cells, suggesting that these RNAs function as important mediators of positive-strand synthesis. Using kinetic labeling experiments, we show that the full-length and subgenomic-length replicative form RNAs rapidly accumulate and then saturate with label, suggesting that the subgenomic-length negative strands are the principal mediators of positive-strand synthesis. Using cycloheximide, which preferentially inhibits negative-strand and to a lesser extent positive-strand synthesis, we demonstrate that cycloheximide treatment equally inhibits full-length and subgenomic-length negative-strand synthesis. Importantly, following treatment, previously transcribed negative strands remain in transcriptionally active complexes even in the absence of new negative-strand synthesis. These findings indicate that the subgenomic-length negative strands are the principal templates of positive-strand synthesis during MHV infection.

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Crystal structure of Senecavirus A 3C protease

10.1101/2020.02.21.959106 ◽

2020 ◽

Author(s):

Kaiwen Meng ◽

Lijie Zhang ◽

Geng Meng

Keyword(s):

Crystal Structure ◽

Structural Biology ◽

Rna Viruses ◽

Rna Replication ◽

Host Cells ◽

Age Group ◽

Virus Maturation ◽

3C Protease ◽

Cleavage Efficiency ◽

Binding Groove

AbstractSenecavirus A (SVA), an emerging picornavirus in porcine population, could infect porcines of all age group and cause FMD-like symptoms. Picornaviridae, a group of RNA viruses do harm to both human and stocks; however, most of picornaviruses are lack of effective vaccines and drugs. Picornaviral 3C protease (3Cpro), as an important role in virus maturation, they basically take charge of poly-protein cleavaging, RNA replication, and multiple interventions on host cells. In this study, we successfully solved the crystal structure of 3Cpro at 1.9 Å resolution. The results showed several differences of the binding groove within picornaviral 3Cpro, and prompted that the accommodate ability of the pocket may associate with the cleavage efficiency. The further research on 3Cpro cleavage efficiency based on structural biology, will prospectively provide an instruction on designing of efficient 3Cpro for universally proteolysis in picornaviral VLP production.

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Host proteostasis modulates influenza evolution

eLife ◽

10.7554/elife.28652 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 12

Author(s):

Angela M Phillips ◽

Luna O Gonzalez ◽

Emmanuel E Nekongo ◽

Anna I Ponomarenko ◽

Sean M McHugh ◽

...

Keyword(s):

Rna Viruses ◽

Rna Virus ◽

Viral Evolution ◽

Virus Evolution ◽

Critical Force ◽

Host Cells ◽

Mutation Rates ◽

Biophysical Properties ◽

And Function ◽

Protein Variants

Predicting and constraining RNA virus evolution require understanding the molecular factors that define the mutational landscape accessible to these pathogens. RNA viruses typically have high mutation rates, resulting in frequent production of protein variants with compromised biophysical properties. Their evolution is necessarily constrained by the consequent challenge to protein folding and function. We hypothesized that host proteostasis mechanisms may be significant determinants of the fitness of viral protein variants, serving as a critical force shaping viral evolution. Here, we test that hypothesis by propagating influenza in host cells displaying chemically-controlled, divergent proteostasis environments. We find that both the nature of selection on the influenza genome and the accessibility of specific mutational trajectories are significantly impacted by host proteostasis. These findings provide new insights into features of host–pathogen interactions that shape viral evolution, and into the potential design of host proteostasis-targeted antiviral therapeutics that are refractory to resistance.

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