Enoxacin Shows Broad-Spectrum Antiviral Activity against Diverse Viruses by Enhancing Antiviral RNA Interference in Insects.

RNA interference (RNAi) functions as the major host antiviral defense in insects, while less is understood about how to utilize antiviral RNAi in controlling viral infection in insects. Enoxacin belongs to the family of synthetic antibacterial compounds based on a fluoroquinolone skeleton that has been previously found to enhance RNAi in mammalian cells. In this study, we show that enoxacin efficiently inhibited viral replication of Drosophila C virus (DCV) and cricket paralysis virus (CrPV) in cultured Drosophila cells. Enoxacin promoted the loading of Dicer-2-processed virus-derived small interfering RNA (siRNA) into the RNA-induced silencing complex, thereby enhancing the antiviral RNAi response in infected cells. Moreover, enoxacin treatment elicited RNAi-dependent in vivo protective efficacy against DCV or CrPV challenge in adult fruit flies. In addition, enoxacin also inhibited the replication of flaviviruses, including dengue virus and Zika virus, in Aedes mosquito cells in an RNAi-dependent manner. Together, our findings demonstrate that enoxacin can enhance RNAi in insects, and enhancing RNAi by enoxacin is an effective antiviral strategy against diverse viruses in insects, which may be exploited as a broad-spectrum antiviral agent to control the vector transmission of arboviruses or viral diseases in insect farming. IMPORTANCE RNAi has been widely recognized as one of the most broadly acting and robust antiviral mechanisms in insects. However, the application of antiviral RNAi in controlling viral infections in insects is less understood. Enoxacin is a fluoroquinolone compound that was previously found to enhance RNAi in mammalian cells, while its RNAi-enhancing activity has not been assessed in insects. Here, we show that enoxacin treatment inhibited viral replication of DCV and CrPV in Drosophila cells and adult fruit flies. Enoxacin promoted the loading of Dicer-generated virus-derived siRNA into the Ago2-incorporated RNA-induced silencing complex and in turn strengthened the antiviral RNAi response in the infected cells. Moreover, enoxacin displayed effective RNAi-dependent antiviral effects against flaviviruses, such as dengue virus and Zika virus, in mosquito cells. This study is the first to demonstrate that enhancing RNAi by enoxacin elicits potent antiviral effects against diverse viruses in insects.

Pesticide-Virus Interactions in Honey Bees: Challenges and Opportunities for Understanding Drivers of Bee Declines.

Honey bees are key agricultural pollinators, but beekeepers continually suffer high annual colony losses owing to a number of environmental stressors, including inadequate nutrition, pressures from parasites and pathogens, and exposure to a wide variety of pesticides. In this review, we examine how two such stressors, pesticides and viruses, may interact in additive or synergistic ways to affect honey bee health. Despite what appears to be a straightforward comparison, there is a dearth of studies examining this issue likely owing to the complexity of such interactions. Such complexities include the wide array of pesticide chemical classes with different modes of actions, the coupling of many bee viruses with ectoparasitic Varroa mites, and the intricate social structure of honey bee colonies. Together, these issues pose a challenge to researchers examining the effects pesticide-virus interactions at both the individual and colony level.

Effect of 1,3-1,6 ß-Glucan on Natural and Experimental Deformed Wing Virus Infection in Newly Emerged Honeybees (Apis mellifera ligustica).

The Western Honeybee is a key pollinator for natural as well as agricultural ecosystems. In the last decade massive honeybee colony losses have been observed worldwide, the result of a complex syndrome triggered by multiple stress factors, with the RNA virus Deformed Wing Virus (DWV) and the mite Varroa destructor playing crucial roles. The mite supports replication of DWV to high titers, which exert an immunosuppressive action and correlate with the onset of the disease. The aim of this study was to investigate the effect of 1,3-1,6 ß-glucan, a natural innate immune system modulator, on honeybee response to low-titer natural and high-titer experimental DWV infection. As the effects exerted by ß-glucans can be remarkably different, depending on the target organism and the dose administered, two parallel experiments were performed, where 1,3-1,6 ß-glucan at a concentration of 0.5% and 2% respectively, was added to the diet of three cohorts of newly emerged honeybees, which were sampled from a Varroa-free apiary and harboured a low endogenous DWV viral titer. Each cohort was subjected to one of the following experimental treatments: no injection, injection of a high-copy number DWV suspension into the haemocel (experimental DWV infection) or injection of PBS into the haemocoel (physical injury). Control bees fed a ß-glucan-free diet were subjected to the same treatments. Viral load, survival rate, haemocyte populations and phenoloxidase activity of each experimental group were measured and compared. The results indicated that oral administration of 0.5% ß-glucan to naturally infected honeybees was associated with a significantly decrease of the number of infected bees and viral load they carried, and with a significant increase of the survival rate, suggesting that this natural immune modulator molecule might contribute to increase honeybee resistance to viral infection.

Antiviral drug valacyclovir treatment combined with a clean feeding system enhances the suppression of salivary gland hypertrophy in laboratory colonies of Glossina pallidipes.

BACKGROUND: Hytrosaviridae cause salivary gland hypertrophy (SGH) syndrome in some infected tsetse flies (Diptera: Glossinidae). Infected male and female G. pallidipes with SGH have a reduced fecundity and fertility. Due to the deleterious impact of the virus on G. pallidipes colonies, adding the antiviral drug valacyclovir to the blood diet and changing the feeding regime to a clean feeding system (each fly receives for each feeding a fresh clean blood meal) have been investigated to develop virus management strategies. Although both approaches used alone successfully reduced the virus load and the SGH prevalence in small experimental groups, considerable time was needed to obtain the desired SGH reduction and both systems were only demonstrated with colonies that had a low initial virus prevalence (SGH ≤ 10%). As problems with SGH are often only recognized once the incidence is already high, it was necessary to demonstrate that this combination would also work for high prevalence colonies. FINDINGS: Combining both methods at colony level successfully suppressed the SGH in G. pallidipes colonies that had a high initial virus prevalence (average SGH of 24%). Six months after starting the combined treatment SGH symptoms were eliminated from the treated colony, in contrast to 28 months required to obtain the same results using clean feeding alone and 21 months using antiviral drug alone. CONCLUSIONS: Combining valacyclovir treatment with the clean feeding system provides faster control of SGH in tsetse than either method alone and is effective even when the initial SGH prevalence is high.

Transgenerational transmission of the Glossina pallidipes hytrosavirus depends on the presence of a functional symbiome.

The vertically transmitted endosymbionts (Sodalis glossinidius and Wigglesworthia glossinidia) of the tsetse fly (Diptera: Glossinidae) are known to supplement dietary deficiencies and modulate the reproductive fitness and the defense system of the fly. Some tsetse fly species are also infected with the bacterium, Wolbachia and with the Glossina hytrosavirus (GpSGHV). Laboratory-bred G. pallidipes exhibit chronic asymptomatic and acute symptomatic GpSGHV infection, with the former being the most common in these colonies. However, under as yet undefined conditions, the asymptomatic state can convert to the symptomatic state, leading to detectable salivary gland hypertrophy (SGH(+)) syndrome. In this study, we investigated the interplay between the bacterial symbiome and GpSGHV during development of G. pallidipes by knocking down the symbionts with antibiotic. Intrahaemocoelic injection of GpSGHV led to high virus titre (10(9) virus copies), but was not accompanied by either the onset of detectable SGH(+), or release of detectable virus particles into the blood meals during feeding events. When the F1 generations of GpSGHV-challenged mothers were dissected within 24 h post-eclosion, SGH(+) was observed to increase from 4.5% in the first larviposition cycle to >95% in the fourth cycle. Despite being sterile, these F1 SGH(+) progeny mated readily. Removal of the tsetse symbiome, however, suppressed transgenerational transfer of the virus via milk secretions and blocked the ability of GpSGHV to infect salivary glands of the F1 progeny. Whereas GpSGHV infects and replicates in salivary glands of developing pupa, the virus is unable to induce SGH(+) within fully differentiated adult salivary glands. The F1 SGH(+) adults are responsible for the GpSGHV-induced colony collapse in tsetse factories. Our data suggest that GpSGHV has co-evolved with the tsetse symbiome and that the symbionts play key roles in the virus transmission from mother to progeny.

Reduction in deformed wing virus infection in larval and adult honey bees (Apis mellifera L.) by double-stranded RNA ingestion.

Deformed wing virus (DWV) is a serious pathogen of the honey bee, Apis mellifera L., vectored by the parasitic mite Varroa destructor. The virus is associated with wing deformity in symptomatic bees, and premature death and reduced colony performance in asymptomatic bees. In the present study we reduced DWV infection by feeding both first instar larvae and adult A. mellifera with a double-stranded (ds) RNA construct, DWV-dsRNA, which is specific to DWV in DWV-inoculated bees, by mixing it with their food. We showed that feeding DWV to larvae causes wing deformity in adult bees in the absence of varroa mites and decreases survival rates of adult bees relative to bees not fed DWV. Feeding larvae with DWV-dsRNA in advance of inoculation with virus reduced the DWV viral level and reduced wing deformity relative to larvae fed DWV or DWV with green fluorescent protein-dsRNA (probably a result of RNA silencing), but did not affect survival to the adult stage. Feeding DWV-dsRNA did not affect larval survival rates, which suggests that dsRNA is non-toxic to larvae. Feeding adult workers with DWV-dsRNA in advance of inoculation with virus increased their longevity and reduced DWV concentration relative to controls.

Coenzyme Q plays opposing roles on bacteria/fungi and viruses in Drosophila innate immunity.

Coenzyme Q (CoQ or ubiquinone) is a lipid-soluble component of virtually all types of cell membranes and has been shown to play multiple metabolic functions. Several clinical diseases including encephalomyopathy, cerebellar ataxia and isolated myopathy were shown to be associated with CoQ deficiency. However, the role of CoQ in immunity has not been defined. In the present study, we showed that flies defective in CoQ biosynthetic gene coq2 were more susceptible to bacterial and fungal infections, while were more resistant to viruses. We found that Drosophila contained both CoQ9 and CoQ10, and food supplement of CoQ10 could partially rescue the impaired immune functions of coq2 mutants. Surprisingly, wild-type flies fed CoQ10 became more susceptible to viral infection, which suggested that extra caution should be taken when using CoQ10 as a food supplement. We further showed that CoQ was essential for normal induction of anti-microbial peptides and amplification of viruses. Our work determined CoQ content in Drosophila and described its function in immunity for the first time.

Small compounds targeted to subunit interfaces arrest maturation in a nonenveloped, icosahedral animal virus.

Nudaurelia omega capensis virus (N omega V) capsids were previously characterized in two morphological forms, a T=4, 485-A-diameter round particle with large pores and a tightly sealed 395-A icosahedrally shaped particle with the same quasi-symmetric surface lattice. The large particle converts to the smaller particle when the pH is lowered from 7.6 to 5, and this activates an autocatalytic cleavage of the viral subunit at residue 570. Here we report that both 1-anilino-8 naphthalene sulfonate (ANS) and the covalent attachment of the thiol-reactive fluorophore, maleimide-ANS (MIANS), inhibit the structural transition and proteolysis at the lower pH. When ANS is exhaustively washed from the particles, the maturation proceeds normally; however, MIANS-modified particles are still inhibited after the same washing treatment, indicating that covalent attachment targets MIANS to a critical location for inhibition. Characterization of the low-pH MIANS product by electron cryo-microscopy (cryo-EM) and image reconstruction demonstrated a morphology intermediate between the two forms previously characterized. A pseudoatomic model of the intermediate configuration was generated by rigid body refinement of the X-ray structure of the subunits (previously determined in the assembled capsid) into the cryo-EM density, allowing a quantitative description of the inhibited intermediate and a hypothesis for the mechanism of the inhibition.

Sensitivity of Invertebrate iridescent virus 6 to organic solvents, detergents, enzymes and temperature treatment.

The sensitivity of Invertebrate iridescent virus 6 (IIV-6) to a selection of organic solvents, detergents, enzymes and heat treatment was assayed in Spodoptera frugiperda (Sf9) cells and by injection of inoculum into larvae of Galleria mellonella. In several cases, the degree of sensitivity of the virus depended on the method of assay; cell culture assays indicated greater losses of activity than insect bioassay. IIV-6 was sensitive to chloroform but sensitivity to ether was only detected by cell culture assay. Sensitivity (defined as a reduction of at least 1 log activity) was detected following treatment by 1 and 0.1% SDS, 1% Triton-X100, 70% ethanol, 70% methanol, 1% sodium deoxycholate, pH 11.1 and 3.0. No sensitivity was detected to 1% Tween 80, 1 M MgCl2, 100 mM EDTA, lipase, phospholipase A2, proteinase K, or trypsin at the concentrations tested. Viral activity was reduced by approximately 4 logs following heating to 70 degrees C for 60 min or 80 degrees C for 30 min. The above observations highlight the need for studies on the role of the virus lipid component in the process of particle entry into cells, and may explain why vertebrate and invertebrate iridoviruses have been reported to differ in their sensitivity to organic solvents and enzymes.

Stimulation of polydnavirus replication by 20-hydroxyecdysone.

During oviposition the endoparasitic wasp Campoletis sonorensis, introduces a polydnavirus into parasitized insects where viral gene expression is required for endoparasite survival. The polydnavirus is integrated into wasp chromosomal DNA and replicates only in the ovary. Ecdysteroids regulate the developmental expression of many insect genes and may regulate polydnavirus replication. Direct verification of viral replication was performed by dot blot hybridization and by amplifying DNA sequences containing the viral integration site; this 'junction' fragment cannot be amplified from integrated virus. Thoracic ligations and in vitro ecdysteroid treatments of wasp ovaries support the hypothesis that polydnavirus DNA replication is regulated by ecdysteroid during parasite development.

Categorizing some early and late transcripts directed by the Autographa californica nuclear polyhedrosis virus.

Using an S1 mapping assay on RNA from Spodoptera frugiperda cells infected by the Autographa californica nuclear polyhedrosis virus in the presence and absence of cycloheximide and aphidicolin, we can distinguish three classes of transcripts. First, there are those whose synthesis is blocked by the DNA synthesis inhibitor aphidicolin and which are therefore late transcripts. These include the late transcript of the 39K gene and a late leftward transcript across the XhoI site in the HindIII-F region. Second, there are those whose synthesis is not blocked by aphidicolin, but whose accumulation is inhibited by the protein synthesis inhibitor cycloheximide and which are therefore presumably delayed early genes. These include the p26 transcript(s), the early 39K transcript and the alpha 2 transcript in the HindIII-K/Q region. Third, there are those whose accumulation is not affected or is enhanced by cycloheximide. These are not necessarily immediate early transcripts, but their response to cycloheximide is clearly different from that of those in the second class. They include the alpha 1 and alpha 3 transcripts in the HindIII-K/Q region and the early leftward transcript across the XhoI site in the HindIII-F region.

Ecdysteroids increase the yield of recombinant protein produced in baculovirus insect cell expression system.

Spodoptera frugiperda cells are the hosts of wild type and recombinant virus in the baculovirus insect cell expression system. The expression of the foreign gene could be enhanced by the addition of ecdysteroids and increased amount of recombinant protein was secreted into the medium. The time and concentration dependence of this effect was followed in the case of 20-hydroxyecdysone-, makisterone and ecdysone. 20-hydroxyecdysone proved to be the most efficient, producing a three fold increase in the level of recombinant protein secreted into the medium, as it was measured by ELISA. This effect was also confirmed by tracing the L-(35S)methionine incorporation into the gene product. Makisterone was also effective in stimulation, while ecdysone proved to be ineffective.

Effect of cytochalasin D on cell morphology and AcMNPV replication in a Spodoptera frugiperda cell line.

Cytochalasin D (CD) is a specific inhibitor of actin microfilament elongation and has been used to identify actin-dependent cellular processes. In this study we observed the effects of this inhibitor on Autographa californica M nuclear polyhedrosis virus infected and uninfected IPLB-SF-21 cells by electron microscopy. The cytochalasin D-induced morphological effects detected in uninfected cells included lobulate nuclei, double nuclei, long retraction processes, increased zeiosis, more frequent plasma membrane indentations, increased vacuolation, more numerous coated pits and vesicles, filamentous masses in the cytoplasm, and decreased surface microvilli. Observation of infected cells treated with CD revealed that viral morphogenesis was severely affected. Few normal-appearing nucleocapsids were seen in the nucleus, and none were detected in the cytoplasm. Instead, long capsid-like tubular structures appeared juxtaposed to the inner nuclear membrane. Very infrequently sections of these structures contained electron dense material. The center of the nucleus contained electron-dense, spidery-like structures, presumably viral DNA. Normal virus was not observed to bud from the plasma membrane but electron-lucent, coreless-particles were. By 50 hr postinfection occasional polyhedra appeared, but these contained few or no enveloped virions. The intranuclear fibrous masses normally associated with infection were significantly reduced. These observations suggest that viral morphogenesis, especially nucleocapsid assembly, is an actin-dependent process.

Proteins induced by Boolarra virus in Drosophila melanogaster line 1 cells.

In Boolarra virus infected Drosophila line 1 cells five virus-induced proteins were readily detected 20 h postinfection. These were A (103,500), alpha (46,000), beta (40,000), B (9,600), and gamma (6,600) and are similar in molecular weight to those induced in vivo by other nodaviruses. The addition of actinomycin D for the 1st h before the labeling period resulted in the production of all five proteins; but when actinomycin D was introduced for the entire labeling period, the synthesis of the products of RNA1 was greatly reduced. Host functions inhibited by actinomycin D may play a major role in the replication of this virus.

The antiviral spectrum of (E)-5-(2-bromovinyl)-2'-deoxyuridine.

The antiviral activity spectrum of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) is not restricted to herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) but also encompasses several other herpesviruses such as suid herpesvirus type 1 (SHV-1), bovid herpesvirus type 1 (BHV-1), simian varicella virus (SVV), herpesvirus saimiri, herpesvirus platyrrhinae, and the baculovirus Trichoplusia ni multiple nuclear polyhedrosis virus. Other herpesviruses such as herpes simplex virus type 2, cytomegalovirus and Epstein-Barr virus are also inhibited by BVdU, albeit to a lesser extent than HSV-1, VZV, SHV-1, BHV-1 and SVV.

Baculovirus replication: effects of inhibitors of macromolecular synthesis.

A number of inhibitors of DNA, RNA, protein and polyamine synthesis have been used to elucidate the mode of replication of baculoviruses. An overview of the viruses, the antimicrobial inhibitors used, and the effects of the drugs are presented. Although certain inhibitors of protein synthesis and DNA synthesis are useful in determining the program of expression of the viral genome into immediate early, delayed early, late and very late phases of synthesis, few drugs have proved useful as antimicrobial agents. Bromovinyldeoxyuridine (BVDU) is a potent inhibitor of baculovirus replication inhibiting viral DNA synthesis by blocking the virus induced DNA polymerase. Preliminary experiments suggest that BVDU suppresses baculovirus disease in insect larvae.

The gene organisation of a small RNA-containing insect virus: comparison with that of mammalian picornaviruses.

The coding regions of an insect virus, cricket paralysis virus, have been mapped using pactamycin. The results suggest that the genome of this virus functions as a polycistronic mRNA, the structural proteins being encoded by the 5' end of the RNA in an order similar to those of mammalian picornaviruses. High-molecular-weight proteins of unknown function map at the 3' end of the genome.

The effects of S-adenosyl methionine (AdoMet) and its analogues on the control of transcription and translation in vitro of the mRNA products of two cytoplasmic polyhedrosis viruses.

S-Adenosyl methionine (AdoMet) and several structurally related compounds were added to in vitro systems for the synthesis of single-stranded RNA by cytoplasmic polyhedrosis virus (CPV) types 1 and 2. The effects of these compounds were examined on the level of transcription and methylation of the RNA products. Of the compounds tested, five increased the polymerase activity in both viruses, the most effective being the D- and L-stereoisomers of S-adenosyl homocysteine (AdoHcy), and the least effective, adenosine. L-AdoHcy, unlike D-AdoHcy, was also a competitive inhibitor of RNA methylation in the presence of [3H]AdoMet. The different response of both viruses to D- and L-AdoHcy suggests that CPV virions contain at least two functionally distinct sites to which AdoMet, or its analogues, bind. One of these is the transcription control site, while the other is the active site(s) for RNA methylation. CPV RNA synthesised in the presence of the methyl donor AdoMet was more efficiently translated in vitro in a wheat-germ translation system than RNA synthesised in the presence of methylation inhibitors. Type 2 CPV-RNA transcripts had a greater degree of methylation than type 1 CPV transcripts and were more effective in stimulating protein synthesis in the translation system. It seems likely that the allosteric control of CPV polymerase by AdoMet and its analogues, and the methylation of the transcripts, ensures the effective transcription and translation of the CPV genome and the stability of the viral messenger RNA.