Differential gene expression and microRNA profile in corpora allata-corpora cardiaca of Aedes aegypti mosquitoes with weak juvenile hormone signalling.

The corpora allata-corpora cardiaca (CA-CC) is an endocrine gland complex that regulates mosquito development and reproduction through the synthesis of juvenile hormone (JH). Epoxidase (Epox) is a key enzyme in the production of JH. We recently utilized CRISPR/Cas9 to establish an epoxidase-deficient (epox-/-) Aedes aegypti line. The CA from epox-/- mutants do not synthesize epoxidated JH III but methyl farneosate (MF), a weak agonist of the JH receptor, and therefore have reduced JH signalling. Illumina sequencing was used to examine the differences in gene expression between the CA-CC from wild type (WT) and epox-/- adult female mosquitoes. From 18,034 identified genes, 317 were significantly differentially expressed. These genes are involved in many biological processes, including the regulation of cell proliferation and apoptosis, energy metabolism, and nutritional uptake. In addition, the same CA-CC samples were also used to examine the microRNA (miRNA) profiles of epox-/- and WT mosquitoes. A total of 197 miRNAs were detected, 24 of which were differentially regulated in epox-/- mutants. miRNA binding sites for these particular miRNAs were identified using an in silico approach; they target a total of 101 differentially expressed genes. Our results suggest that a lack of epoxidase, besides affecting JH synthesis, results in the diminishing of JH signalling that have significant effects on Ae. aegypti CA-CC transcriptome profiles, as well as its miRNA repertoire.

Aedes aegypti gut transcriptomes respond differently to microbiome transplants from field-caught or laboratory-reared mosquitoes.

The mosquito microbiome is critical for host development and plays a major role in many aspects of mosquito biology. While the microbiome is commonly dominated by a small number of genera, there is considerable variation in composition among mosquito species, life stages, and geography. How the host controls and is affected by this variation is unclear. Using microbiome transplant experiments, we asked whether there were differences in transcriptional responses when mosquitoes of different species were used as microbiome donors. We used microbiomes from four different donor species spanning the phylogenetic breadth of the Culicidae, collected either from the laboratory or the field. We found that when recipients received a microbiome from a donor reared in the laboratory, the response was remarkably similar regardless of donor species. However, when the donor had been collected from the field, many more genes were differentially expressed. We also found that while the transplant procedure did have some effect on the host transcriptome, this is likely to have had a limited effect on mosquito fitness. Overall, our results highlight the possibility that variation in mosquito microbiome communities is associated with variability in host-microbiome interactions and further demonstrate the utility of the microbiome transplantation technique for investigating host-microbe interactions in mosquitoes.

ALKBH8 as a potential N6 -methyladenosine (m6 A) eraser in insects.

The N6 -methyladenosine (m6 A) machinery functions through three groups of proteins in eukaryotic cells, including m6 A writers, erasers and readers. The m6 A cellular machinery has mostly been characterised in mammalian species, and the relevant literature on insects is currently scant. While homologues of m6 A writers and readers have been reported from insects, no erasers have been described so far. Here, using BLAST search, we searched for potential erasers in insects. While we found homologues of human m6 A eraser ALKBH5 in termites, beetles and true bugs, they could not be found in representative dipteran and lepidopteran species. However, a potential m6 A eraser, ALKBH8, was identified and experimentally investigated. Our results showed that ALKBH8 can reduce the m6 A levels of Aedes aegypti and Drosophila melanogaster RNAs, suggesting that AeALKBH8 could be a candidate m6 A eraser in insects.

Altered gene expression profile of Wolbachia pipientis wAlbB strain following transinfection from its native host Aedes albopictus to Aedes aegypti cells.

Wolbachia is an obligate intracellular bacterial symbiont prevalent among arthropods and nematodes. To survive and reproduce, Wolbachia interacts with and modifies host subcellular structures, while sensing and responding to changes within the cellular environment. In mutualistic associations, Wolbachia may provision the host with metabolites, or help to maintain the chemical homeostasis of the host cell. Some strains can rapidly invade insect populations by manipulating host reproductive biology, while also preventing viral replication, allowing their use in vector control of arthropod-borne viruses. The Aedes albopictus-derived strain wAlbB is promising in this regard. When transinfected into the Yellow fever mosquito, Aedes aegypti, wAlbB reaches high frequencies within wild populations, and strongly inhibits viral transmission. Despite its obvious potential, much is still unknown about the molecular interactions between Wolbachia and host that enable its use in vector control. Furthermore, most Wolbachia transinfection research to date has focused on host effects. In the current study, we used a cell line model to explore the effect of transinfection of wAlbB from Ae. albopictus to Ae. aegypti. Using RNA sequencing, we show that several genes associated with host-symbiont interactions were downregulated by transinfection, with the greatest downregulation exhibited by prophage-associated genes.

Transcriptional response of Wolbachia-transinfected Aedes aegypti mosquito cells to dengue virus at early stages of infection.

Mosquito-borne flaviviruses are responsible for viral infections and represent a considerable public health burden. Aedes aegypti is the principal vector of dengue virus (DENV), therefore understanding the intrinsic virus-host interactions is vital, particularly in the presence of the endosymbiont Wolbachia, which blocks virus replication in mosquitoes. Here, we examined the transcriptional response of Wolbachia-transinfected Ae. aegypti Aag2 cells to DENV infection. We identified differentially expressed immune genes that play a key role in the activation of anti-viral defence such as the Toll and immune deficiency pathways. Further, genes encoding cytosine and N6-adenosine methyltransferases and SUMOylation, involved in post-transcriptional modifications, an antioxidant enzyme, and heat-shock response were up-regulated at the early stages of DENV infection and are reported here for the first time. Additionally, several long non-coding RNAs were among the differentially regulated genes. Our results provide insight into Wolbachia-transinfected Ae. aegypti's initial virus recognition and transcriptional response to DENV infection.

Knockout of Dicer-2 in the Sf9 cell line enhances the replication of Spodoptera frugiperda rhabdovirus and conditionally increases baculovirus replication.

The Sf9 cell line, originally isolated from the ovarian tissue of Spodoptera frugiperda larvae, is widely used in academia and industry for the baculovirus-mediated production of recombinant proteins and virus-like particles. RNA interference (RNAi) is a conserved antiviral pathway present in eukaryotic organisms and is the primary antiviral defence mechanism in insects. Recent evidence has implicated RNAi as an antiviral response to baculovirus infection in Sf9 cells. To test this hypothesis, CRISPR/Cas9 technology was used to disable the RNAi pathway in Sf9 cells by knocking out Dicer-2, the protein responsible for cleaving viral double-stranded RNA precursors into short interfering RNAs. Infection of Dicer-2 knockout Sf9 cells with either the wild-type baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV), recombinant AcMNPV (rAcMNPV) expressing ß-galactosidase (ß-gal), or rAcMNPV expressing a wasp venom protein (Vn50) at a multiplicity of infection (m.o.i.) of 1 resulted in a modest increase in virus replication compared to control Sf9 cells under adherent culture conditions. In contrast, Dicer-2 knockout Sf9 monolayer or suspension cultures infected by the rAcMNPV expressing ß-gal at higher m.o.i.s (3.5 and 20) did not exhibit increases in either viral DNA replication or ß-gal production. Intriguingly, during long-term passaging in suspension, Dicer-2 knockout Sf9 cultures underwent transient crashes in cell proliferation and viability. It was discovered that these periods of low growth and viability coincided with a dramatic increase in the RNA levels of S. frugiperda rhabdovirus, a recently identified adventitious virus that persistently infects the Sf9 cell line, suggesting a role for Dicer-2 in managing chronic viral infections in this industrially relevant insect cell line.

Environmental Temperature, but Not Male Age, Affects Wolbachia and Prophage WO Thereby Modulating Cytoplasmic Incompatibility in the Parasitoid Wasp, Habrobracon Hebetor.

Wolbachia is an endosymbiotic bacterium found in many species of arthropods and manipulates its host reproduction. Cytoplasmic incompatibility (CI) is one of the most common manipulations that is induced when an uninfected female mates with a Wolbachia-infected male. The CI factors (cifA and cifB genes) are encoded by phage WO that naturally infects Wolbachia. Here, we questioned whether an environmental factor (temperature) or host factor (male age) affected the strength of the CI phenotype in the ectoparasitoid wasp, Habrobracon hebetor. We found that temperature, but not male age, results in reduced CI penetrance. Consistent with these results, we also found that the expression of the cif CI factors decreased in temperature-exposed males but was consistent across aging male wasps. Similar to studies of other insect systems, cifA showed a higher expression level than cifB, and male hosts showed increased cif expression relative to females. Our results suggest that prophage WO is present in the Wolbachia-infected wasps and expression of cif genes contributes to the induction of CI in this insect. It seems that male aging has no effect on the intensity of CI; however, temperature affects Wolbachia and prophage WO titers as well as expression levels of cif genes, which modulate the CI level.

Two repeated motifs enriched within some enhancers and origins of replication are bound by SETMAR isoforms in human colon cells.

Setmar is a gene specific to simian genomes. The function(s) of its isoforms are poorly understood and their existence in healthy tissues remains to be validated. Here we profiled SETMAR expression and its genome-wide binding landscape in colon tissue. We found isoforms V3 and V6 in healthy and tumour colon tissues as well as incell lines. In two colorectal cell lines SETMAR binds to several thousand Hsmar1 and MADE1 terminal ends, transposons mostly located in non-genic regions of active chromatin including in enhancers. It also binds to a 12-bp motifs similar to an inner motif in Hsmar1 and MADE1 terminal ends. This motif is interspersed throughout the genome and is enriched in GC-rich regions as well as in CpG islands that contain constitutive replication origins. It is also found in enhancers other than those associated with Hsmar1 and MADE1. The role of SETMAR in the expression of genes, DNA replication and in DNA repair are discussed.

Effect of Wolbachia wAlbB on a positive-sense RNA negev-like virus: a novel virus persistently infecting Aedes albopictus mosquitoes and cells.

The Aedes aegypti mosquito is the primary vector of several medically important arboviruses. The endosymbiotic bacterium, Wolbachia pipientis, has emerged as a means of blocking transmission of arboviruses such as dengue and Zika viruses. One Wolbachia strain that has shown potential in field trials is wAlbB, a naturally occurring Wolbachia strain of the Asian tiger mosquito Aedes albopictus. When transinfected into Ae. aegypti, wAlbB exhibits strong virus inhibition. In addition to modulating arboviruses, Wolbachia also modulates some insect-specific viruses. Here, we explored the effect of Wolbachia on the virome of the Ae. albopictus cell line Aa23 naturally infected with wAlbB and also a stably transinfected recipient Ae. aegypti cell line (Aag2.wAlbB). RNA sequencing and bioinformatic analysis on both cell lines revealed an 11 kb genome of a single-stranded positive-sense RNA negev-like virus related to the recently proposed negevirus taxon. We denoted this novel virus as Aedes albopictus negev-like virus (AalNLV). Tetracycline clearance of Wolbachia from Aa23 cells did not significantly affect AalNLV levels, while in Aag2.wAlbB cells, a significant increase in virus genome RNA copies was observed. We further investigated the inhibitory effect of wAlbB on AalNLV and another positive-sense RNA virus, cell fusing agent virus, which is present in Aag2 cells and known to be suppressed by Wolbachia. wAlbB suppressed both viruses, with the effect on AalNLV being more striking. The findings from this study further supplement our understanding of the complex interaction between Wolbachia, host and virome.

Discovery of Novel Crustacean and Cephalopod Flaviviruses: Insights into the Evolution and Circulation of Flaviviruses between Marine Invertebrate and Vertebrate Hosts.

Most described flaviviruses (family Flaviviridae) are disease-causing pathogens of vertebrates maintained in zoonotic cycles between mosquitoes or ticks and vertebrate hosts. Poor sampling of flaviviruses outside vector-borne flaviviruses such as Zika virus and dengue virus has presented a narrow understanding of flavivirus diversity and evolution. In this study, we discovered three crustacean flaviviruses (Gammarus chevreuxi flavivirus, Gammarus pulex flavivirus, and Crangon crangon flavivirus) and two cephalopod flaviviruses (Southern Pygmy squid flavivirus and Firefly squid flavivirus). Bayesian and maximum likelihood phylogenetic methods demonstrate that crustacean flaviviruses form a well-supported clade and share a more closely related ancestor with terrestrial vector-borne flaviviruses than with classical insect-specific flaviviruses. In addition, we identify variants of Wenzhou shark flavivirus in multiple gazami crab (Portunus trituberculatus) populations, with active replication supported by evidence of an active RNA interference response. This suggests that Wenzhou shark flavivirus moves horizontally between sharks and gazami crabs in ocean ecosystems. Analyses of the mono- and dinucleotide composition of marine flaviviruses compared to that of flaviviruses with known host status suggest that some marine flaviviruses share a nucleotide bias similar to that of vector-borne flaviviruses. Furthermore, we identify crustacean flavivirus endogenous viral elements that are closely related to elements of terrestrial vector-borne flaviviruses. Taken together, these data provide evidence of flaviviruses circulating between marine vertebrates and invertebrates, expand our understanding of flavivirus host range, and offer potential insights into the evolution and emergence of terrestrial vector-borne flaviviruses.IMPORTANCE Some flaviviruses are known to cause disease in vertebrates and are typically transmitted by blood-feeding arthropods such as ticks and mosquitoes. While an ever-increasing number of insect-specific flaviviruses have been described, we have a narrow understanding of flavivirus incidence and evolution. To expand this understanding, we discovered a number of novel flaviviruses that infect a range of crustaceans and cephalopod hosts. Phylogenetic analyses of these novel marine flaviviruses suggest that crustacean flaviviruses share a close ancestor to all terrestrial vector-borne flaviviruses, and squid flaviviruses are the most divergent of all known flaviviruses to date. Additionally, our results indicate horizontal transmission of a marine flavivirus between crabs and sharks. Taken together, these data suggest that flaviviruses move horizontally between invertebrates and vertebrates in ocean ecosystems. This study demonstrates that flavivirus invertebrate-vertebrate host associations have arisen in flaviviruses at least twice and may potentially provide insights into the emergence or origin of terrestrial vector-borne flaviviruses.

Wolbachia Endosymbiont of the Horn Fly (Haematobia irritans irritans): a Supergroup A Strain with Multiple Horizontally Acquired Cytoplasmic Incompatibility Genes.

The horn fly, Haematobia irritansirritans, is a hematophagous parasite of livestock distributed throughout Europe, Africa, Asia, and the Americas. Welfare losses on livestock due to horn fly infestation are estimated to cost between $1 billion and $2.5 billion (U.S. dollars) annually in North America and Brazil. The endosymbiotic bacterium Wolbachia pipientis is a maternally inherited manipulator of reproductive biology in arthropods and naturally infects laboratory colonies of horn flies from Kerrville, TX, and Alberta, Canada, but it has also been identified in wild-caught samples from Canada, the United States, Mexico, and Hungary. Reassembly of PacBio long-read and Illumina genomic DNA libraries from the Kerrville H. i. irritans genome project allowed for a complete and circularized 1.3-Mb Wolbachia genome (wIrr). Annotation of wIrr yielded 1,249 coding genes, 34 tRNAs, 3 rRNAs, and 5 prophage regions. Comparative genomics and whole-genome Bayesian evolutionary analysis of wIrr compared to published Wolbachia genomes suggested that wIrr is most closely related to and diverged from Wolbachia supergroup A strains known to infect Drosophila spp. Whole-genome synteny analyses between wIrr and closely related genomes indicated that wIrr has undergone significant genome rearrangements while maintaining high nucleotide identity. Comparative analysis of the cytoplasmic incompatibility (CI) genes of wIrr suggested two phylogenetically distinct CI loci and acquisition of another cifB homolog from phylogenetically distant supergroup A Wolbachia strains, suggesting horizontal acquisition of these loci. The wIrr genome provides a resource for future examination of the impact Wolbachia may have in both biocontrol and potential insecticide resistance of horn flies.IMPORTANCE Horn flies, Haematobia irritans irritans, are obligate hematophagous parasites of cattle having significant effects on production and animal welfare. Control of horn flies mainly relies on the use of insecticides, but issues with resistance have increased interest in development of alternative means of control. Wolbachia pipientis is an endosymbiont bacterium known to have a range of effects on host reproduction, such as induction of cytoplasmic incompatibility, feminization, male killing, and also impacts vector transmission. These characteristics of Wolbachia have been exploited in biological control approaches for a range of insect pests. Here we report the assembly and annotation of the circular genome of the Wolbachia strain of the Kerrville, TX, horn fly (wIrr). Annotation of wIrr suggests its unique features, including the horizontal acquisition of additional transcriptionally active cytoplasmic incompatibility loci. This study provides the foundation for future studies of Wolbachia-induced biological effects for control of horn flies.

Aedes Anphevirus: an Insect-Specific Virus Distributed Worldwide in Aedes aegypti Mosquitoes That Has Complex Interplays with Wolbachia and Dengue Virus Infection in Cells.

Insect-specific viruses (ISVs) of the yellow fever mosquito Aedes aegypti have been demonstrated to modulate transmission of arboviruses such as dengue virus (DENV) and West Nile virus by the mosquito. The diversity and composition of the virome of A. aegypti, however, remains poorly understood. In this study, we characterized Aedes anphevirus (AeAV), a negative-sense RNA virus from the order Mononegavirales AeAV identified from Aedes cell lines was infectious to both A. aegypti and Aedes albopictus cells but not to three mammalian cell lines. To understand the incidence and genetic diversity of AeAV, we assembled 17 coding-complete and two partial genomes of AeAV from available transcriptome sequencing (RNA-Seq) data. AeAV appears to transmit vertically and be present in laboratory colonies, wild-caught mosquitoes, and cell lines worldwide. Phylogenetic analysis of AeAV strains indicates that as the A. aegypti mosquito has expanded into the Americas and Asia-Pacific, AeAV has evolved into monophyletic African, American, and Asia-Pacific lineages. The endosymbiotic bacterium Wolbachia pipientis restricts positive-sense RNA viruses in A. aegypti Reanalysis of a small RNA library of A. aegypti cells coinfected with AeAV and Wolbachia produces an abundant RNA interference (RNAi) response consistent with persistent virus replication. We found Wolbachia enhances replication of AeAV compared to a tetracycline-cleared cell line, and AeAV modestly reduces DENV replication in vitro The results from our study improve understanding of the diversity and evolution of the virome of A. aegypti and adds to previous evidence that shows Wolbachia does not restrict a range of negative-strand RNA viruses.IMPORTANCE The mosquito Aedes aegypti transmits a number of arthropod-borne viruses (arboviruses), such as dengue virus and Zika virus. Mosquitoes also harbor insect-specific viruses that may affect replication of pathogenic arboviruses in their body. Currently, however, there are only a few insect-specific viruses described from A. aegypti in the literature. Here, we characterize a novel negative-strand virus, AeAV. Meta-analysis of A. aegypti samples showed that it is present in A. aegypti mosquitoes worldwide and is vertically transmitted. Wolbachia-transinfected mosquitoes are currently being used in biocontrol, as they effectively block transmission of several positive-sense RNA viruses in mosquitoes. Our results demonstrate that Wolbachia enhances the replication of AeAV and modestly reduces dengue virus replication in a cell line model. This study expands our understanding of the virome in A. aegypti as well as providing insight into the complexity of the Wolbachia virus restriction phenotype.

The major oligosaccharyl transferase complex genes are not involved in dengue virus replication in Aedes aegypti mosquitoes.

Replication of the dengue virus (DENV) genome occurs in a vesicle in the endoplasmic reticulum by a complex of host and viral proteins. Two host proteins, STT3A and STT3B, as members of the oligosaccharyl transferase complex, have been implicated in playing structural roles in the vesicle in mammalian cells, and the absence of these proteins has been shown to decrease DENV replication. Aedes aegypti is the main vector of the virus and has been used previously as a model organism to study mosquito-virus interactions. In this study, we found that genes of the oligosaccharyl transferase complex have no effect on replication of DENV in mosquito cells.

Wolbachia induce cytoplasmic incompatibility and affect mate preference in Habrobracon hebetor to increase the chance of its transmission to the next generation.

Wolbachia are common intracellular bacteria that are generally found in arthropods, including a high proportion of insects and also some nematodes. This intracellular symbiont can affect sex ratio with a variety of reproductive anomalies in the host, including cytoplasmic incompatibility (CI) in haplodiploids. In this study, we questioned if the parasitoid wasp, Habrobracon hebetor (Hym.: Braconidae), an important biological control agent of many lepidopteran larvae, is infected with Wolbachia. To test this, DNA was extracted from adult insects and subjected to PCR using specific primers to Wolbachia target genes. The results showed a high rate of Wolbachia infection in this parasitoid wasp. To determine the biological function of Wolbachia in H. hebetor, we removed this bacterium from the wasps using antibiotic treatment (cured wasps). Results of crossing experiments revealed that Wolbachia induced CI in H. hebetor in which cured females crossed with infected males produced only males, while both male and female progeny were observed for other crosses. Also, we showed that the presence of Wolbachia in females increased fecundity and female offspring of this parasitoid wasp. The presence of Wolbachia in the males had no significant effect on fecundity and female production, but might have incurred costs. We also investigated the effect of Wolbachia on mate choice and found that Wolbachia affects mating behavior of H. hebetor. Together, we showed that Wolbachia induces CI in H. hebetor and affects host mating behavior in favor of its transmission. Wolbachia utilize these strategies to increase the frequency of infected females in the host population.

Drosophila microRNA modulates viral replication by targeting a homologue of mammalian cJun.

MicroRNAs (miRNAs) are important regulators of biological processes, including host-virus interaction. This study investigated the involvement of Drosophila melanogaster miR-8-5p in host-virus interaction. Drosophila flies and cells challenged with Drosophila C virus (DCV) were found to have lower miR-8-5p abundance compared to uninfected samples. Lowering miR-8-5p abundance by experimental inhibition of the miRNA led to an increase in viral accumulation, suggesting that the observed decrease in the miR-8-5p abundance during DCV infection enhances viral replication. miR-8-5p putative targets were identified and included dJun, a transcription factor gene whose mammalian homologue cJun is induced by various viruses through kinase activation. Increasing miR-8-5p abundance using miR-8-5p mimics resulted in a decrease in dJun and GFP reporter levels. Furthermore, when the putative target in dJun was mutated, addition of miR-8-5p mimics did not result in the same antagonistic effect on dJun. These results show negative regulation of dJun by miR-8-5p and suggest that an miRNA-mediated pathway is involved in dJun regulation during viral infection. To analyse the role of dJun during DCV infection, dJun was knocked down in cells prior to DCV infection. Knockdown of dJun decreased DCV replication, providing evidence that dJun up-regulation that is concomitant with miR-8-5p down-regulation during DCV infection supports viral replication. These results highlight the role of miRNA in regulating the transcription factor gene dJun and uncover a previously unrecognized mechanism by which dJun is regulated during host-virus interaction.

ICTV Virus Taxonomy Profile: Ascoviridae.

The family Ascoviridae includes viruses with circular dsDNA genomes of 100-200 kbp characterized by oblong enveloped virions of 200-400 nm in length. Ascoviruses mainly infect lepidopteran larvae and are mechanically transmitted by parasitoid wasps in which they may also replicate. Most known members belong to the genus Ascovirus, except one virus, that of the genus Toursvirus, which replicates in both its lepidopteran and parasitoid vector hosts. Ascoviruses cause high mortality among economically important insect pests, thereby controlling insect populations. This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Ascoviridae, which is available at www.ictv.global/report/ascoviridae.

Understanding the role of microRNAs in the interaction of Aedes aegypti mosquitoes with an insect-specific flavivirus.

The Flavivirus genus contains some of the most prevalent vector-borne viruses, such as the dengue, Zika and yellow fever viruses that cause devastating diseases in humans. However, the insect-specific clade of flaviviruses is restricted to mosquito hosts, albeit they have retained the general features of the genus, such as genome structure and replication. The interactions between insect-specific flaviviruses (ISFs) and their mosquito hosts are largely unknown. Pathogenic flaviviruses are known to modulate host-derived microRNAs (miRNAs), a class of non-coding RNAs that are important in controlling gene expression. Alterations in miRNAs may represent changes in host gene expression and promote understanding of virus-host interactions. The role of miRNAs in ISF-mosquito interactions is largely unknown. A recently discovered Australian ISF, Palm Creek virus (PCV), has the ability to suppress medically relevant flaviviruses. Here, we investigated the potential involvement of miRNAs in PCV infection using the model mosquito Aedes aegypti. By combining small-RNA sequencing and bioinformatics analysis, differentially expressed miRNAs were determined. Our results indicated that PCV infection hardly affects host miRNAs. Out of 101 reported miRNAs of Ae. aegypti, only aae-miR-2940-5p had a significantly altered expression over the course of infection. However, further analysis of aae-miR-2940-5p revealed that this miRNA does not have any direct impact on PCV replication in vitro. Thus, overall the results suggest that PCV infection has a limited effect on the mosquito miRNA profile and therefore miRNAs may not play a significant role in the PCV-Ae. aegypti interaction.

Discovery of new orbiviruses and totivirus from Anopheles mosquitoes in Eastern Australia.

Three new viruses classifiable within the Totivirus and Orbivirus genera were detected from Anopheles mosquito species collected in Eastern Australia. The viruses could not be isolated in C6/36 mosquito cell cultures but were shown to replicate in their mosquito hosts by small RNA analysis. The viruses grouped phylogenetically with other viruses recently detected in insects. These discoveries contribute to a better understanding of commensal viruses in Australian mosquitoes and the evolution of these viruses.

MicroRNA-like viral small RNA from Dengue virus 2 autoregulates its replication in mosquito cells.

MicroRNAs (miRNAs) are small regulatory RNAs that play significant roles in most cellular processes. In the seemingly endless arms race between hosts and pathogens, viruses also encode miRNAs that facilitate successful infection. In search of functional miRNAs or viral small RNAs (vsRNAs) encoded by Dengue virus (DENV), deep sequencing data of virus-infected Aedes aegypti mosquitoes were used. From six vsRNAs, with candidate stem-loop structures in the 5' and 3' untranslated regions of the viral genomic RNA, inhibition of DENV-vsRNA-5 led to significant increases in viral replication. Silencing of RNA interference (RNAi)/miRNA pathways' associated proteins showed that Argonaute 2 is mainly involved in DENV-vsRNA-5 biogenesis. Cloning of the precursor stem loop, immunoprecipitations, ectopic expression and detection in RNAi-deficient C6/36, and the mammalian Vero cell lines further confirmed DENV-vsRNA-5 production. Furthermore, significant impact of synthetic mimic and inhibitor of DENV-vsRNA-5 on DENV RNA levels revealed DENV-vsRNA-5's role in virus autoregulation by targeting the virus nonstructural protein 1 gene. Notably, DENV-vsRNA-5 homologous mimics from DENV serotypes 1 and 4, but not 3, inhibited DENV-2 replication. The results revealed that DENV is able to encode functional vsRNAs, and one of those, which resembles miRNAs, specifically targets a viral gene, opening an avenue for possible utilization of the small RNA to limit DENV replication.