Molecular basis of RNA recombination in the 3'UTR of chikungunya virus genome.

Chikungunya virus (CHIKV) is a rapidly spreading re-emergent virus transmitted from mosquitoes to humans. The emergence of epidemic variants has been associated with changes in the viral genome, such as the duplication of repeated sequences in the 3' untranslated region (UTR). Indeed, blocks of repeated sequences seemingly favor RNA recombination, providing the virus with a unique ability to continuously change the 3'UTR architecture during host switching. In this work, we provide experimental data on the molecular mechanism of RNA recombination and describe specific sequence and structural elements in the viral 3'UTR that favor template switching of the viral RNA-dependent RNA polymerase on the 3'UTR. Furthermore, we found that a 3'UTR deletion mutant that exhibits markedly delayed replication in mosquito cells and impaired transmission in vivo, recombines in reference laboratory strains of mosquitoes. Altogether, our data provide novel experimental evidence indicating that RNA recombination can act as a nucleic acid repair mechanism to add repeated sequences that are associated to high viral fitness in mosquito during chikungunya virus replication.

Detection and quantification of natural Wolbachia in Aedes aegypti in Metropolitan Manila, Philippines using locally designed primers.

Background: The Philippines bears health and economic burden caused by high dengue cases annually. Presently, the Philippines still lack an effective and sustainable vector management. The use of Wolbachia, a maternally transmitted bacterium, that mitigate arbovirus transmission has been recommended. Cytoplasmic incompatibility and viral blocking, two characteristics that make Wolbachia suitable for vector control, depend on infection prevalence and density. There are no current Wolbachia release programs in the Philippines, and studies regarding the safety of this intervention. Here, we screened for Wolbachia in Aedes aegypti collected from Metropolitan Manila, Philippines. We designed location-specific primers for qPCR to test whether this improved Wolbachia detection in Ae. aegypti. We explored if host sex and Wolbachia strain could be potential factors affecting Wolbachia density. Methods: Ae. aegypti mosquitoes (n=429) were screened for natural Wolbachia by taqman qPCR using location-specific Wolbachia surface protein primers (wspAAML) and known 16S rRNA primers. Samples positive for wspAAML (n=267) were processed for Sanger sequencing. We constructed a phylogenetic tree using IQ-TREE 2 to further characterize Wolbachia present in the Philippine Ae. aegypti. We then compared Wolbachia densities between Wolbachia groups and host sex. Statistical analyses were done using GraphPad Prism 9.0. Results: Wolbachia prevalence for 16S rRNA (40%) and wspAAML (62%) markers were high. Wolbachia relative densities for 16S rRNA ranged from -3.84 to 2.71 and wspAAML from -4.02 to 1.81. Densities were higher in male than female mosquitoes. Wolbachia strains detected in Ae. aegypti clustered into supergroup B. Some 54% (123/226) of these sequences clustered under a group referred to here as "wAegML," that belongs to the supergroup B, which had a significantly lower density than wAegB/wAlbB, and wAlbA strains. Conclusion: Location-specific primers improved detection of natural Wolbachia in Ae. aegypti and allowed for relative quantification. Wolbachia density is relatively low, and differed between host sexes and Wolbachia strains. An economical way of confirming sporadic or transient Wolbachia in Ae. aegypti is necessary while considering host sex and bacterial strain.

No detectable fitness cost of infection by cell-fusing agent virus in Aedes aegypti mosquitoes.

Aedes mosquitoes are well-known vectors of arthropod-borne viruses (arboviruses). Mosquitoes are more frequently infected with insect-specific viruses (ISVs) that cannot infect vertebrates. Some ISVs interfere with arbovirus replication in mosquito vectors, which has gained attention for potential use against arbovirus transmission. Cell-fusing agent virus (CFAV), a widespread ISV, can reduce arbovirus dissemination in Ae. aegypti. However, vectorial capacity is largely governed by other parameters than pathogen load, including mosquito survival and biting behaviour. Understanding how ISVs impact these mosquito fitness-related traits is critical to assess the potential risk of using ISVs as biological agents. Here, we examined the effects of CFAV infection on Ae. aegypti mosquito fitness. We found no significant reduction in mosquito survival, blood-feeding behaviour and reproduction, suggesting that Ae. aegypti is tolerant to CFAV. The only detectable effect was a slight increase in human attraction of CFAV-infected females in one out of eight trials. Viral tolerance is beneficial for introducing CFAV into natural mosquito populations, whereas the potential increase in biting activity must be further investigated. Our results provide the first insight into the link between ISVs and Aedes mosquito fitness and highlight the importance of considering all aspects of vectorial capacity for arbovirus control using ISVs.

In vitro characterization of cell-fusing agent virus DNA forms in Aedes aegypti mosquitoes.

How non-retroviral endogenous viral elements (EVEs) are established is a long-standing question. Viral DNA (vDNA) forms of RNA viruses are likely to be EVE precursors. Cell-fusing agent virus (CFAV) is a major insect-specific virus (ISV) in the Aedes aegypti mosquitoes and one of the few existing non-retroviral RNA viruses found as EVEs. We characterized CFAV-derived vDNA in the cell line to understand the mechanism of why current viruses are rarely endogenized. vDNA production was affected by cell culture media independent of CFAV replication. vDNAs that correspond to different regions covering the entire viral genome were detected, implying multiple initiation sites exist. A considerable proportion of vDNAs corresponded to ssDNA. Higher vDNA copies were detected in the cytoplasm than the nucleus. Our findings provide valuable insights into the intracellular characteristics of ISV-derived vDNAs, which will aid in understanding the underlying mechanisms of non-retroviral EVE formation.

Wolbachia-induced inhibition of O'nyong nyong virus in Anopheles mosquitoes is mediated by Toll signaling and modulated by cholesterol.

Enhanced host immunity and competition for metabolic resources are two main competing hypotheses for the mechanism of Wolbachia-mediated pathogen inhibition in arthropods. Using an Anopheles mosquito - somatic Wolbachia infection - O'nyong nyong virus (ONNV) model, we demonstrate that the mechanism underpinning Wolbachia-mediated virus inhibition is up-regulation of the Toll innate immune pathway. However, the viral inhibitory properties of Wolbachia were abolished by cholesterol supplementation. This result was due to Wolbachia-dependent cholesterol-mediated suppression of Toll signaling rather than competition for cholesterol between Wolbachia and virus. The inhibitory effect of cholesterol was specific to Wolbachia-infected Anopheles mosquitoes and cells. These data indicate that both Wolbachia and cholesterol influence Toll immune signaling in Anopheles mosquitoes in a complex manner and provide a functional link between the host immunity and metabolic competition hypotheses for explaining Wolbachia-mediated pathogen interference in mosquitoes. In addition, these results provide a mechanistic understanding of the mode of action of Wolbachia-induced pathogen blocking in Anophelines, which is critical to evaluate the long-term efficacy of control strategies for malaria and Anopheles-transmitted arboviruses.

Intracellular Interactions Between Arboviruses and Wolbachia in Aedes aegypti.

Aedes aegypti is inherently susceptible to arboviruses. The geographical expansion of this vector host species has led to the persistence of Dengue, Zika, and Chikungunya human infections. These viruses take advantage of the mosquito's cell to create an environment conducive for their growth. Arboviral infection triggers transcriptomic and protein dysregulation in Ae. aegypti and in effect, host antiviral mechanisms are compromised. Currently, there are no existing vaccines able to protect human hosts from these infections and thus, vector control strategies such as Wolbachia mass release program is regarded as a viable option. Considerable evidence demonstrates how the presence of Wolbachia interferes with arboviruses by decreasing host cytoskeletal proteins and lipids essential for arboviral infection. Also, Wolbachia strengthens host immunity, cellular regeneration and causes the expression of microRNAs which could potentially be involved in virus inhibition. However, variation in the magnitude of Wolbachia's pathogen blocking effect that is not due to the endosymbiont's density has been recently reported. Furthermore, the cellular mechanisms involved in this phenotype differs depending on Wolbachia strain and host species. This prompts the need to explore the cellular interactions between Ae. aegypti-arboviruses-Wolbachia and how different Wolbachia strains overall affect the mosquito's cell. Understanding what happens at the cellular and molecular level will provide evidence on the sustainability of Wolbachia vector control.

Defective viral genomes as therapeutic interfering particles against flavivirus infection in mammalian and mosquito hosts.

Arthropod-borne viruses pose a major threat to global public health. Thus, innovative strategies for their control and prevention are urgently needed. Here, we exploit the natural capacity of viruses to generate defective viral genomes (DVGs) to their detriment. While DVGs have been described for most viruses, identifying which, if any, can be used as therapeutic agents remains a challenge. We present a combined experimental evolution and computational approach to triage DVG sequence space and pinpoint the fittest deletions, using Zika virus as an arbovirus model. This approach identifies fit DVGs that optimally interfere with wild-type virus infection. We show that the most fit DVGs conserve the open reading frame to maintain the translation of the remaining non-structural proteins, a characteristic that is fundamental across the flavivirus genus. Finally, we demonstrate that the high fitness DVG is antiviral in vivo both in the mammalian host and the mosquito vector, reducing transmission in the latter by up to 90%. Our approach establishes the method to interrogate the DVG fitness landscape, and enables the systematic identification of DVGs that show promise as human therapeutics and vector control strategies to mitigate arbovirus transmission and disease.

Defective viral genomes from chikungunya virus are broad-spectrum antivirals and prevent virus dissemination in mosquitoes.

Defective viral genomes (DVGs) are truncated and/or rearranged viral genomes produced during virus replication. Described in many RNA virus families, some of them have interfering activity on their parental virus and/or strong immunostimulatory potential, and are being considered in antiviral approaches. Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes spp. that infected millions of humans in the last 15 years. Here, we describe the DVGs arising during CHIKV infection in vitro in mammalian and mosquito cells, and in vivo in experimentally infected Aedes aegypti mosquitoes. We combined experimental and computational approaches to select DVG candidates most likely to have inhibitory activity and showed that, indeed, they strongly interfere with CHIKV replication both in mammalian and mosquito cells. We further demonstrated that some DVGs present broad-spectrum activity, inhibiting several CHIKV strains and other alphaviruses. Finally, we showed that pre-treating Aedes aegypti with DVGs prevented viral dissemination in vivo.

Evidence For Long-Lasting Transgenerational Antiviral Immunity in Insects.

Transgenerational immune priming (TGIP) allows memory-like immune responses to be transmitted from parents to offspring in many invertebrates. Despite increasing evidence for TGIP in insects, the mechanisms involved in the transfer of information remain largely unknown. Here, we show that Drosophila melanogaster and Aedes aegypti transmit antiviral immunological memory to their progeny that lasts throughout generations. We observe that TGIP, which is virus and sequence specific but RNAi independent, is initiated by a single exposure to disparate RNA viruses and also by inoculation of a fragment of viral double-stranded RNA. The progeny, which inherit a viral DNA that is only a fragment of the viral RNA used to infect the parents, display enriched expression of genes related to chromatin and DNA binding. These findings represent a demonstration of TGIP for RNA viruses in invertebrates, broadly increasing our understanding of the immune response, host genome plasticity, and antiviral memory of the germline.

Non-retroviral Endogenous Viral Element Limits Cognate Virus Replication in Aedes aegypti Ovaries.

Endogenous viral elements (EVEs) are viral sequences integrated in host genomes. A large number of non-retroviral EVEs was recently detected in Aedes mosquito genomes, leading to the hypothesis that mosquito EVEs may control exogenous infections by closely related viruses. Here, we experimentally investigated the role of an EVE naturally found in Aedes aegypti populations and derived from the widespread insect-specific virus, cell-fusing agent virus (CFAV). Using CRISPR-Cas9 genome editing, we created an Ae. aegypti line lacking the CFAV EVE. Absence of the EVE resulted in increased CFAV replication in ovaries, possibly modulating vertical transmission of the virus. Viral replication was controlled by targeting of viral RNA by EVE-derived P-element-induced wimpy testis-interacting RNAs (piRNAs). Our results provide evidence that antiviral piRNAs are produced in the presence of a naturally occurring EVE and its cognate virus, demonstrating a functional link between non-retroviral EVEs and antiviral immunity in a natural insect-virus interaction.

Differential Small RNA Responses against Co-Infecting Insect-Specific Viruses in Aedes albopictus Mosquitoes.

The mosquito antiviral response has mainly been studied in the context of arthropod-borne virus (arbovirus) infection in female mosquitoes. However, in nature, both female and male mosquitoes are frequently infected with insect-specific viruses (ISVs). ISVs are capable of infecting the reproductive organs of both sexes and are primarily maintained by vertical transmission. Since the RNA interference (RNAi)-mediated antiviral response plays an important antiviral role in mosquitoes, ISVs constitute a relevant model to study sex-dependent antiviral responses. Using a naturally generated viral stock containing three distinct ISVs, Aedes flavivirus (AEFV), Menghai rhabdovirus (MERV), and Shinobi tetra virus (SHTV), we infected adult Aedes albopictus females and males and generated small RNA libraries from ovaries, testes, and the remainder of the body. Overall, both female and male mosquitoes showed unique small RNA profiles to each co-infecting ISV regardless of the sex or tissue tested. While all three ISVs generated virus-derived siRNAs, only MERV generated virus-derived piRNAs. We also studied the expression of PIWI genes in reproductive tissues and carcasses. In contrast to Piwi5-9, Piwi1-4 were abundantly expressed in ovaries and testes, suggesting that Piwi5-9 are involved in exogenous viral piRNA production. Together, our results show that ISV-infected Aedes albopictus produce viral small RNAs in a virus-specific manner and that male mosquitoes mount a similar small RNA-mediated antiviral response to that of females.

RNA recombination at Chikungunya virus 3'UTR as an evolutionary mechanism that provides adaptability.

The potential of RNA viruses to adapt to new environments relies on their ability to introduce changes in their genomes, which has resulted in the recent expansion of re-emergent viruses. Chikungunya virus is an important human pathogen transmitted by mosquitoes that, after 60 years of exclusive circulation in Asia and Africa, has rapidly spread in Europe and the Americas. Here, we examined the evolution of CHIKV in different hosts and uncovered host-specific requirements of the CHIKV 3'UTR. Sequence repeats are conserved at the CHIKV 3'UTR but vary in copy number among viral lineages. We found that these blocks of repeated sequences favor RNA recombination processes through copy-choice mechanism that acts concertedly with viral selection, determining the emergence of new viral variants. Functional analyses using a panel of mutant viruses indicated that opposite selective pressures in mosquito and mammalian cells impose a fitness cost during transmission that is alleviated by recombination guided by sequence repeats. Indeed, drastic changes in the frequency of viral variants with different numbers of repeats were detected during host switch. We propose that RNA recombination accelerates CHIKV adaptability, allowing the virus to overcome genetic bottlenecks within the mosquito host. These studies highlight the role of 3'UTR plasticity on CHIKV evolution, providing a new paradigm to explain the significance of sequence repetitions.

Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing.

Cas9-mediated gene editing is a powerful tool for addressing research questions in arthropods. Current approaches rely upon delivering Cas9 ribonucleoprotein (RNP) complex by embryonic microinjection, which is challenging, is limited to a small number of species, and is inefficient even in optimized taxa. Here we develop a technology termed Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) to deliver Cas9 RNP to the arthropod germline by injection into adult female mosquitoes. We identify a peptide (P2C) that mediates transduction of Cas9 RNP from the female hemolymph to the developing mosquito oocytes, resulting in heritable gene editing of the offspring with efficiency as high as 0.3 mutants per injected mosquito. We demonstrate that P2C functions in six mosquito species. Identification of taxa-specific ovary-specific ligand-receptor pairs may further extend the use of ReMOT Control for gene editing in novel species.

Uncovering the Repertoire of Endogenous Flaviviral Elements in Aedes Mosquito Genomes.

Endogenous viral elements derived from nonretroviral RNA viruses have been described in various animal genomes. Whether they have a biological function, such as host immune protection against related viruses, is a field of intense study. Here, we investigated the repertoire of endogenous flaviviral elements (EFVEs) in Aedes mosquitoes, the vectors of arboviruses such as dengue and chikungunya viruses. Previous studies identified three EFVEs from Aedes albopictus cell lines and one from Aedes aegypti cell lines. However, an in-depth characterization of EFVEs in wild-type mosquito populations and individual mosquitoes in vivo has not been performed. We detected the full-length DNA sequence of the previously described EFVEs and their respective transcripts in several A. albopictus and A. aegypti populations from geographically distinct areas. However, EFVE-derived proteins were not detected by mass spectrometry. Using deep sequencing, we detected the production of PIWI-interacting RNA-like small RNAs, in an antisense orientation, targeting the EFVEs and their flanking regions in vivo The EFVEs were integrated in repetitive regions of the mosquito genomes, and their flanking sequences varied among mosquito populations. We bioinformatically predicted several new EFVEs from a Vietnamese A. albopictus population and observed variation in the occurrence of those elements among mosquitoes. Phylogenetic analysis of an A. aegypti EFVE suggested that it integrated prior to the global expansion of the species and subsequently diverged among and within populations. The findings of this study together reveal the substantial structural and nucleotide diversity of flaviviral integrations in Aedes genomes. Unraveling this diversity will help to elucidate the potential biological function of these EFVEs.IMPORTANCE Endogenous viral elements (EVEs) are whole or partial viral sequences integrated in host genomes. Interestingly, some EVEs have important functions for host fitness and antiviral defense. Because mosquitoes also have EVEs in their genomes, characterizing these EVEs is a prerequisite for their potential use to manipulate the mosquito antiviral response. In the study described here, we focused on EVEs related to the Flavivirus genus, to which dengue and Zika viruses belong, in individual Aedes mosquitoes from geographically distinct areas. We show the existence in vivo of flaviviral EVEs previously identified in mosquito cell lines, and we detected new ones. We show that EVEs have evolved differently in each mosquito population. They produce transcripts and small RNAs but not proteins, suggesting a function at the RNA level. Our study uncovers the diverse repertoire of flaviviral EVEs in Aedes mosquito populations and contributes to an understanding of their role in the host antiviral system.

Factors influencing infection and transmission of Anopheles gambiae densovirus (AgDNV) in mosquitoes.

Anopheles gambiae densovirus (AgDNV) is a potential microbial agent for paratransgenesis and gene transduction in An. gambiae, the major vector of human malaria in sub-Saharan Africa. Understanding the interaction between AgDNV and An. gambiae is critical for using AgDNV in a basic and applied manner for Anopheles gene manipulation. Here, we tested the effects of mosquito age, sex, blood feeding status, and potential for horizontal transmission using an enhanced green fluorescent protein (EGFP) reporter AgDNV system. Neither mosquito age at infection nor feeding regime affected viral titers. Female mosquitoes were more permissive to viral infection than males. Despite low viral titers, infected males were able to venereally transmit virus to females during mating, where the virus was localized with the transferred sperm in the spermathecae. These findings will be useful for designing AgDNV-based strategies to manipulate Anopheles gambiae.

Bunyaviruses are common in male and female Ixodes scapularis ticks in central Pennsylvania.

The blacklegged tick Ixodes scapularis is widely distributed in the United States and transmits multiple pathogens to humans, wildlife and domestic animals. Recently, several novel viruses in the family Bunyaviridae (South Bay virus (SBV) and Blacklegged tick phlebovirus (BTPV)) were identified infecting female I. scapularis ticks collected in New York State. We used metagenomic sequencing to investigate the distribution of viruses infecting male and female I. scapularis ticks collected in Centre County, Pennsylvania. We identified both SBV and BTPV in both male and female ticks from all collection locations. The role of male I. scapularis in pathogen epidemiology has been overlooked because they rarely bite and are not considered important pathogen vectors. However, males may act as reservoirs for pathogens that can then be transmitted to females during mating. Our data highlight the importance of examining all potential avenues of pathogen maintenance and transmission throughout the vector-pathogen life cycle in order to understand the epidemiology of tick-borne pathogens.

In vitro and in vivo host range of Anopheles gambiae densovirus (AgDNV).

AgDNV is a powerful gene transduction tool and potential biological control agent for Anopheles mosquitoes. Using a GFP reporter virus system, we investigated AgDNV host range specificity in four arthropod cell lines (derived from An. gambiae, Aedes albopictus and Drosophila melanogaster) and six mosquito species from 3 genera (An. gambiae, An. arabiensis, An. stephensi, Ae. albopictus, Ae. aegypti and Culex tarsalis). In vitro, efficient viral invasion, replication and GFP expression was only observed in MOS55 An. gambiae cells. In vivo, high levels of GFP were observed in An. gambiae mosquitoes. Intermediate levels of GFP were observed in the closely related species An. arabiensis. Low levels of GFP were observed in An. stephensi, Ae. albopictus, Ae. aegypti and Cx. tarsalis. These results suggest that AgDNV is a specific gene transduction tool for members of the An. gambiae species complex, and could be potentially developed into a biocontrol agent with minimal off-target effects.

Anopheles gambiae densovirus (AgDNV) has negligible effects on adult survival and transcriptome of its mosquito host.

Mosquito densoviruses (DNVs) are candidate agents for paratransgenic control of malaria and other vector-borne diseases. Unlike other mosquito DNVs, the Anopheles gambiae DNV (AgDNV) is non-pathogenic to larval mosquitoes. However, the cost of infection upon adults and the molecular mechanisms underpinning infection in the mosquito host are unknown. Using life table analysis, we show that AgDNV infection has minimal effects on An. gambiae survival (no significant effect in 2 replicates and a slight 2 day survival decrease in the third replicate). Using microarrays, we show that AgDNV has very minimal effect on the adult mosquito transcriptome, with only 4-15 genes differentially regulated depending on the statistical criteria imposed. The minimal impact upon global transcription provides some mechanistic understanding of lack of virus pathogenicity, suggesting a long co-evolutionary history that has shifted towards avirulence. From an applied standpoint, lack of strong induced fitness costs makes AgDNV an attractive agent for paratransgenic malaria control.

Native microbiome impedes vertical transmission of Wolbachia in Anopheles mosquitoes.

Over evolutionary time, Wolbachia has been repeatedly transferred between host species contributing to the widespread distribution of the symbiont in arthropods. For novel infections to be maintained, Wolbachia must infect the female germ line after being acquired by horizontal transfer. Although mechanistic examples of horizontal transfer exist, there is a poor understanding of factors that lead to successful vertical maintenance of the acquired infection. Using Anopheles mosquitoes (which are naturally uninfected by Wolbachia) we demonstrate that the native mosquito microbiota is a major barrier to vertical transmission of a horizontally acquired Wolbachia infection. After injection into adult Anopheles gambiae, some strains of Wolbachia invade the germ line, but are poorly transmitted to the next generation. In Anopheles stephensi, Wolbachia infection elicited massive blood meal-induced mortality, preventing development of progeny. Manipulation of the mosquito microbiota by antibiotic treatment resulted in perfect maternal transmission at significantly elevated titers of the wAlbB Wolbachia strain in A. gambiae, and alleviated blood meal-induced mortality in A. stephensi enabling production of Wolbachia-infected offspring. Microbiome analysis using high-throughput sequencing identified that the bacterium Asaia was significantly reduced by antibiotic treatment in both mosquito species. Supplementation of an antibiotic-resistant mutant of Asaia to antibiotic-treated mosquitoes completely inhibited Wolbachia transmission and partly contributed to blood meal-induced mortality. These data suggest that the components of the native mosquito microbiota can impede Wolbachia transmission in Anopheles. Incompatibility between the microbiota and Wolbachia may in part explain why some hosts are uninfected by this endosymbiont in nature.