Enoxacin shows a broad-spectrum antiviral activity against diverse viruses by enhancing antiviral RNAi 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 showed 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 siRNA into the RNA-induced silencing complex, thereby enhancing antiviral RNAi response in infected cells. Moreover, enoxacin treatment elicited an RNAi-dependent in vivo protective efficacy against DCV or CrPV challenge in adult fruit flies. In addition, enoxacin also inhibited replication of flaviviruses, including Dengue virus and Zika virus, in Aedes mosquito cells in an RNAi-dependent manner. Together, our findings demonstrated 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 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 mechanism in insects. However, the application of antiviral RNAi in controlling viral infections in insects is less understood. Enoxacin is a fluoroquinolone compound that has been previously found to enhance RNAi in mammalian cells, while its RNAi-enhancing activity has not been assessed in insects. Herein, we showed that enoxacin treatment inhibited viral replication of DCV and CrPV in Drosophila cells and in adult fruit flies. Enoxacin promoted the loading of Dicer-generated virus-derived siRNA into Ago2-incorporated RNA-induced silencing complex, and in turn strengthened the antiviral RNAi response in the infected cells. Moreover, enoxacin also 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 efficacies against diverse viruses in insects.

Loquacious Modulates Flaviviral RNA Replication in Mosquito Cells

Arthropod-borne viruses infect both mosquito and mammalian hosts. While much is known about virus-host interactions that modulate viral gene expression in their mammalian host, much less is known about the interactions that involve inhibition, subversion or avoidance strategies in the mosquito host. A novel RNA-Protein interaction detection assay was used to detect proteins that directly or indirectly bind to dengue viral genomes in infected mosquito cells. Membrane-associated mosquito proteins SEC61A1 and Loquacious (Loqs) were found to be in complex with the viral RNA. Depletion analysis demonstrated that both SEC61A1 and Loqs have pro-viral functions in the dengue viral infectious cycle. Co-localization and pull-down assays showed that Loqs interacts with viral protein NS3 and both full-length and subgenomic viral RNAs. While Loqs coats the entire positive-stranded viral RNA, it binds selectively to the 3’ end of the negative-strand of the viral genome. In-depth analyses showed that the absence of Loqs did not affect translation or turnover of the viral RNA but modulated viral replication. Loqs also displayed pro-viral functions for several flaviviruses in infected mosquito cells, suggesting a conserved role for Loqs in flavivirus-infected mosquito cells.Author SummaryThere is a wealth of information that dictates virus-host interactions in flavivirus-infected mammalian cells, yet there is only sparse information on the mechanisms that modulate viral gene expression in the mosquito host. Using a novel RNA-protein detection assay, the interactions of SEC61A1 and Loqs with the dengue viral genome were found to have proviral functions in infected mosquito cells. In particular, Loqs forms complexes with the positive-strand of the viral RNA and the very 3’ end of the negative-strand viral RNA. Further analyses showed that Loqs modulates viral RNA replication of dengue virus and gene amplification of several other flaviviral genomes. These findings argue that Loqs is an essential proviral host factor in mosquitos.

Flavivirus infections induce a Golgi stress response in vertebrate and mosquito cells

The stress of the Golgi apparatus is an autoregulatory mechanism that is induced to compensate for greater demand in the Golgi functions. No examples of Golgi stress responses due to physiological stimuli are known. Furthermore, the impact on this organelle of viral infections that occupy the vesicular transport during replication is unknown. In this work, we evaluated if a Golgi stress response is triggered during dengue and Zika viruses replication, two flaviviruses whose replicative cycle is heavily involved with the Golgi complex, in vertebrate and mosquito cells. Using GM-130 as a Golgi marker, and treatment with monensin as a positive control for the induction of the Golgi stress response, a significant expansion of the Golgi cisternae was observed in BHK-21, Vero E6 and mosquito cells infected with either virus. Activation of the TFE3 pathway was observed in the infected cells as indicated by the translocation from the cytoplasm to the nucleus of TFE3 and increased expression of pathway targeted genes. Of note, no sign of activation of the stress response was observed in CRFK cells infected with Feline Calicivirus (FCV), a virus released by cell lysis, not requiring vesicular transport. Finally, dilatation of the Golgi complex and translocation of TFE3 was observed in vertebrate cells expressing dengue and Zika viruses NS1, but not NS3. These results indicated that infections by dengue and Zika viruses induce a Golgi stress response in vertebrate and mosquito cells due to the increased demand on the Golgi complex imposed by virion and NS1 processing and secretion.

Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos

Mosquito-borne diseases present a worldwide public health burden. Current efforts to understand and counteract them have been aided by the use of cultured mosquito cells. Moreover, application in mammalian cells of forward genetic approaches such as CRISPR screens have identified essential genes and genes required for host-pathogen interactions, and in general, aided in functional annotation of genes. An equivalent approach for genetic screening of mosquito cell lines has been lacking. To develop such an approach, we design a new bioinformatic portal for sgRNA library design in several mosquito genomes, engineer mosquito cell lines to express Cas9 and accept sgRNA at scale, and identify optimal promoters for sgRNA expression in several mosquito species. We then optimize a recombination-mediated cassette exchange system to deliver CRISPR sgRNA and perform pooled CRISPR screens in an Anopheles cell line. Altogether, we provide a platform for high-throughput genome-scale screening in cell lines from disease vector species.

Monitoring Mitochondrial Function in Aedes albopictus C6/36 Cell Line during Dengue Virus Infection

Aedes aegypti and Aedes albopictus mosquitoes are responsible for dengue virus (DENV) transmission in tropical and subtropical areas worldwide, where an estimated 3 billion people live at risk of DENV exposure. DENV-infected individuals show symptoms ranging from sub-clinical or mild to hemorrhagic fever. Infected mosquitoes do not show detectable signs of disease, even though the virus maintains a lifelong persistent infection. The interactions between viruses and host mitochondria are crucial for virus replication and pathogenicity. DENV infection in vertebrate cells modulates mitochondrial function and dynamics to facilitate viral proliferation. Here, we describe that DENV also regulates mitochondrial function and morphology in infected C6/36 mosquito cells (derived from Aedes albopictus). Our results showed that DENV infection increased ROS (reactive oxygen species) production, modulated mitochondrial transmembrane potential and induced changes in mitochondrial respiration. Furthermore, we offer the first evidence that DENV causes translocation of mitofusins to mitochondria in the C6/36 mosquito cell line. Another protein Drp-1 (Dynamin-related protein 1) did not localize to mitochondria in DENV-infected cells. This observation therefore ruled out the possibility that the abovementioned alterations in mitochondrial function are associated with mitochondrial fission. In summary, this report provides some key insights into the virus–mitochondria crosstalk in DENV infected mosquito cells.

Enhancement of alphavirus replication in mammalian cells at sub-physiological temperatures

Chikungunya virus (CHIKV) is a re-emerging Alphavirus transmitted by Aedes mosquitoes and causing fever, rash and arthralgia. Currently there are no vaccines or antiviral agents against CHIKV, therefore it is important to understand the molecular details of CHIKV replication. In this regard, the function of the Alphavirus non-structural protein 3 (nsP3) remains enigmatic. Building on previous studies (Gao et al, 2019), we generated a panel of mutants in a conserved and surface exposed cluster in the nsP3 alphavirus unique domain (AUD) and tested their replication phenotype using a sub-genomic replicon (SGR) in mammalian and mosquito cells. We identified three mutants that replicated poorly in mammalian cells but showed no defect in mosquito cells. We further showed that these mutants were temperature-sensitive, rather than species-specific, as they exhibited no replication defect in mammalian cells at sub-physiological temperature (28°C). Similar effects were observed in the context of infectious CHIKV as well as a closely related virus: O’Nyong Nyong virus (ONNV). Intriguingly, this analysis also revealed that the wildtype SGR replicated much more efficiently at sub-physiological temperature as compared to 37°C. This was not due to impaired interferon responses as this enhancement was also observed in Vero cells. Neither was this due to a defect in the phosphorylation of eIF2α as treatment with ISRIB, an inhibitor of global translation attenuation, did not compensate for replication defects at 37°C. However, we noticed significant differences between the sizes and numbers of virus-induced stress granules (SG) at physiological and sub-physiological temperatures. As cells in the periphery will be at sub-physiological temperatures, and these will be the first cells infected in the mammalian host following a mosquito bite, we propose that alphaviruses have evolved mechanisms to limit antiviral responses in these cells to promote viral genome replication.Author summaryChikungunya virus (CHIKV) is a re-emerging arbovirus. It is transmitted by Aedes species of mosquitos and poses massive epidemic threats. Arboviruses are unique in that they must be able to replicate efficiently in both the mosquito vector and the mammalian host, and therefore at different temperatures. Importantly, the first cells infected in the mammalian host following a mosquito bite will be in the skin and therefore at sub-physiological temperature (lower than 37°C). Here we show that mutants within one of the CHIKV proteins (nsP3) were unable to replicate at 37°C but replicated efficiently in mammalian cells at a sub-physiological temperature (28°C). We also showed that the wildtype virus replicated more efficiently at 28°C in comparison to at 37°C in mammalian cells. We investigated the mechanism behind this phenomenon and showed that at sub-physiological temperatures the virus induced the formation of smaller and more numerous cytoplasmic stress granules. We propose that alphaviruses have evolved mechanisms to promote their replication in mammalian cells at sub-physiological temperatures to facilitate infection of mammals via a cutaneous mosquito bite.

Experimental Evolution of West Nile Virus at Higher Temperatures Facilitates Broad Adaptation and Increased Genetic Diversity

West Nile virus (WNV, Flaviviridae, Flavivirus) is a mosquito-borne flavivirus introduced to North America in 1999. Since 1999, the Earth’s average temperature has increased by 0.6 °C. Mosquitoes are ectothermic organisms, reliant on environmental heat sources. Temperature impacts vector–virus interactions which directly influence arbovirus transmission. RNA viral replication is highly error-prone and increasing temperature could further increase replication rates, mutation frequencies, and evolutionary rates. The impact of temperature on arbovirus evolutionary trajectories and fitness landscapes has yet to be sufficiently studied. To investigate how temperature impacts the rate and extent of WNV evolution in mosquito cells, WNV was experimentally passaged 12 times in Culex tarsalis cells, at 25 °C and 30 °C. Full-genome deep sequencing was used to compare genetic signatures during passage, and replicative fitness was evaluated before and after passage at each temperature. Our results suggest adaptive potential at both temperatures, with unique temperature-dependent and lineage-specific genetic signatures. Further, higher temperature passage was associated with significantly increased replicative fitness at both temperatures and increases in nonsynonymous mutations. Together, these data indicate that if similar selective pressures exist in natural systems, increases in temperature could accelerate emergence of high-fitness strains with greater phenotypic plasticity.

Morphological Description of Den-3 Virus Infection Cells through EDTA Blood Feed In Aedes Aegypti Mosquitoe

Mosquito Ae. aegypti is the main dengue virus vector which causes the virus to grow and develop properly. The Artificial Membrane Feeding (AMF) method is a method of indirectly transmitting the dengue virus in the Ae. aegypti. This method uses blood feed with EDTA anticoagulant and DEN-3 virus in the insectarium. One way to detect the dengue virus found in Ae mosquito cells. aegypti is an immunohistochemical Streptavidin Biotin Peroxidase Complex (SBPC). In this method, the morphological picture of DEN-3 virus infection cells will be seen through the heparin anticoagulant blood feed using the AMF method. Anticoagulants function to slow the blood clotting to feed the Ae mosquitoes. Aegypti. The aim of this study was to determine the Positive Infection Rate of DEN-3 virus and the morphological picture of DEN-3 virus infection cells using the AMF method of EDTA anticoagulant blood feed through SBPC immunocytochemical examination of Ae mosquitoes. aegypti. The design method of this study was experimental, infected Ae.aegypti mosquitoes using anticoagulant blood containing DEN-3 virus orally as an infectious sample. Negative control used Culex Sp mosquitoes and positive control used adult Ae.aegypti mosquitoes which were infected with DEN-3 virus by injection. Detection of DEN-3 virus in Ae.aegypti mosquitoes through SBPC immunositochemical examination. Results of Positive Infection Rate for Ae.aegypti mosquitoes with EDTA anticoagulant as much as 23.20% through SBPC immunocytochemical examination with a picture of cell morphology of DEN-3 virus infection. The conclusion of SBPC immunositochemical examination showed positive cell morphology of DEN-3 virus infection with EDTA anticoagulant in Ae.aegypti mosquitoes fed by human blood orally through the AMF method.

Development of genus-specific universal primers for the detection of flaviviruses

Background Flaviviruses are representative arboviruses carried by arthropods and/or vertebrates; these viruses can pose a public health concern in many countries. By contrast, it is known that a novel virus group called insect-specific flaviviruses (ISFs) also infects arthropods, although no such virus has yet been isolated from vertebrates. The characteristics of ISFs, which affect replication of human-pathogenic flaviviruses within co-infected mosquito cells or mosquitoes without affecting the mosquitoes themselves, mean that we should pay attention to both ISFs and human-pathogenic flaviviruses, despite the fact that ISFs appear not to be directly hazardous to human health. To assess the risk of diseases caused by flaviviruses, and to better understand their ecology, it is necessary to know the extent to which flaviviruses are harbored by arthropods. Methods We developed a novel universal primer for use in a PCR-based system to detect a broad range of flaviviruses. We then evaluated its performance. The utility of the novel primer pair was evaluated in a PCR assay using artificially synthesized oligonucleotides derived from a template viral genome sequence. The utility of the primer pair was also examined by reverse transcription PCR (RT-PCR) using cDNA templates prepared from virus-infected cells or crude supernatants prepared from virus-containing mosquito homogenates. Results The novel primer pair amplified the flavivirus NS5 sequence (artificially synthesized) in all samples tested (six species of flavivirus that can cause infectious diseases in humans, and flaviviruses harbored by insects). In addition, the novel primer pair detected viral genomes in cDNA templates prepared from mosquito cells infected with live flavivirus under different infectious conditions. Finally, the viral genome was detected with high sensitivity in crude supernatants prepared from pooled mosquito homogenates. Conclusion This PCR system based on a novel primer pair makes it possible to detect arthropod-borne flaviviruses worldwide (the primer pair even detected viruses belonging to different genetic subgroups). As such, an assay based on this primer pair may help to improve public health and safety, as well as increase our understanding of flavivirus ecology.

Characterization of a novel reassortment Tibet orbivirus isolated from Culicoides spp. in Yunnan, PR China

Orbiviruses are arboviruses with 10 double-stranded linear RNA segments, and some have been identified as pathogens of dramatic epizootics in both wild and domestic ruminants. Tibet orbivirus (TIBOV) is a new orbivirus isolated from hematophagous insects in recent decades, and, currently, most of the strains have been isolated from insects in PR China, except for two from Japan. In this study, we isolated a novel reassortment TIBOV strain, YN15-283-01, from Culicoides spp. To identify and understand more characteristics of YN15-283-01, electrophoresis profiles of the viral genome, electron microscopic observations, plaque assays, growth curves in various cell lines, and bioinformatic analysis were conducted. The results indicated that YN15-283-01 replicated efficiently in mosquito cells, rodent cells and several primate cells. Furthermore, the maximum likelihood phylogenetic trees and simplot analysis of the 10 segments indicated that YN15-283-01 is a natural reassortment isolate that had emerged mainly from XZ0906 and SX-2017a.