Arthropod-borne viruses (arboviruses) persist in a natural cycle that includes infections of humans or other vertebrates and transmission between vertebrates by infected arthropods, most commonly ...mosquitos. Arboviruses can cause serious, sometimes fatal diseases in humans and other vertebrates but cause little pathology in their mosquito vectors. Knowledge of the interactions between mosquito vectors and the arboviruses that they transmit is an important facet of developing schemes to control transmission. Mosquito innate immune responses to virus infection modulate virus replication in the vector, and understanding the components and mechanisms of the immune response could lead to improved methods for interrupting the transmission cycle. The most important aspect of mosquito antiviral defense is the exogenous small interfering RNA (exo-siRNA) pathway, one arm of the RNA interference (RNAi) silencing response. Our research as well as that of many other groups over the past 25 years to define this pathway are reviewed here. A more recently recognized but less well-understood RNA-mediated mosquito defense against arbovirus infections, the PIWI-interacting RNA (piRNA) pathway, is also described.
Mosquito-borne arboviruses cause serious diseases in humans that are increasingly becoming public health problems, yet arbovirus infections cause minimal pathology in the mosquito vector, allowing ...persistent infections and lifelong virus transmission. The principal mosquito innate immune response to virus infections, RNAi, differs substantially from the human immune response and this difference could be the basis for the disparate outcomes of infection in the two hosts. Understanding the mosquito antiviral immune response could lead to strategies for interruption of arbovirus transmission and greatly reduce disease. Research focused on RNAi as the primary mosquito antiviral response has the greatest potential for developing a full understanding of mosquito innate immunity. This article reviews our current knowledge of mosquito antiviral RNAi and charts some of the future directions needed to fill knowledge gaps.
RNA interference (RNAi) was shown over 18 years ago to be a mechanism by which arbovirus replication and transmission could be controlled in arthropod vectors. During the intervening period, research ...on RNAi has defined many of the components and mechanisms of this antiviral pathway in arthropods, yet a number of unexplored questions remain. RNAi refers to RNA-mediated regulation of gene expression. Originally, the term described silencing of endogenous genes by introduction of exogenous double-stranded (ds)RNA with the same sequence as the gene to be silenced. Further research has shown that RNAi comprises three gene regulation pathways that are mediated by small RNAs: the small interfering (si)RNA, micro (mi)RNA, and Piwi-interacting (pi)RNA pathways. The exogenous (exo-)siRNA pathway is now recognized as a major antiviral innate immune response of arthropods. More recent studies suggest that the piRNA and miRNA pathways might also have important roles in arbovirus-vector interactions. This review will focus on current knowledge of the role of the exo-siRNA pathway as an arthropod vector antiviral response and on emerging research into vector piRNA and miRNA pathway modulation of arbovirus-vector interactions. Although it is assumed that arboviruses must evade the vector's antiviral RNAi response in order to maintain their natural transmission cycles, the strategies by which this is accomplished are not well defined. RNAi is also an important tool for arthropod gene knock-down in functional genomics studies and in development of arbovirus-resistant mosquito populations. Possible arbovirus strategies for evasion of RNAi and applications of RNAi in functional genomics analysis and arbovirus transmission control will also be reviewed.
The P-element-induced wimpy testis (PIWI)-associated RNA (piRNA) pathway is known for its role in the protection of genome integrity in the germline of
by silencing transposable elements. The piRNAs ...that target transposons originate from piRNA clusters in transposon-rich regions of the
genome and are processed by three PIWI family proteins. In
and
mosquitoes, which are two of the most important vectors of arthropod-borne viruses (arboviruses), the number of PIWI family genes has expanded and some are expressed in somatic, as well as germline, tissues. These discoveries have led to active research to explore the possible expanded functional roles of the piRNA pathway in vector mosquitoes. Virus genome-derived piRNAs (which will be referred to as (virus name) vpiRNAs) have been demonstrated in
spp. cultured cells and mosquitoes after infection by arthropod-borne alpha-, bunya-, and flaviviruses. However, although
also is an important arbovirus vector and has an expansion of PIWI family genes, vpiRNAs have seldom been documented in this genus after arbovirus infection. Generation of complementary DNA (cDNA) fragments from RNA genomes of alpha-, bunya-, and flaviviruses (viral-derived cDNAs, vDNAs) has been demonstrated in cultured
spp. cells and mosquitoes, and endogenous viral elements (EVEs), cDNA fragments of non-retroviral RNA virus genomes, are found more abundantly in genomes of
and
than other vector mosquitoes. These observations have led to speculation that vDNAs are integrated into vector genomes to form EVEs, which serve as templates for the transcription of antiviral vpiRNA precursors. However, no EVEs derived from alphavirus genomes have been demonstrated in genomes of any vector mosquito. In addition, although EVEs have been shown to be a source of piRNAs, the preponderance of EVEs described in
spp. vectors are more closely related to the genomes of persistently infecting insect-specific viruses than to acutely infecting arboviruses. Furthermore, the signature patterns of the "ping-pong" amplification cycle that maintains transposon-targeting piRNAs in
are also evident in alphavirus and bunyavirus vpiRNAs, but not in vpiRNAs of flaviviruses. These divergent observations have rendered deciphering the mechanism(s) of biogenesis and potential role of vpiRNAs in the mosquito-arbovirus arms race difficult, and the focus of this review will be to assemble major findings regarding vpiRNAs and antiviral immunity in the important arbovirus vectors from
and
genera.
Many viral pathogens cycle between humans and insects. These viruses must have evolved strategies for rapid adaptation to different host environments. However, the mechanistic basis for the ...adaptation process remains poorly understood. To study the mosquito-human adaptation cycle, we examined changes in RNA structures of the dengue virus genome during host adaptation. Deep sequencing and RNA structure analysis, together with fitness evaluation, revealed a process of host specialization of RNA elements of the viral 3'UTR. Adaptation to mosquito or mammalian cells involved selection of different viral populations harvesting mutations in a single stem-loop structure. The host specialization of the identified RNA structure resulted in a significant viral fitness cost in the non-specialized host, posing a constraint during host switching. Sequence conservation analysis indicated that the identified host adaptable stem loop structure is duplicated in dengue and other mosquito-borne viruses. Interestingly, functional studies using recombinant viruses with single or double stem loops revealed that duplication of the RNA structure allows the virus to accommodate mutations beneficial in one host and deleterious in the other. Our findings reveal new concepts in adaptation of RNA viruses, in which host specialization of RNA structures results in high fitness in the adapted host, while RNA duplication confers robustness during host switching.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. ...Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The exogenous RNA interference (RNAi) pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (si)RNAs are key components of this pathway. ...Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2)-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2) cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV) corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Flavivirus genus includes a large number of medically relevant pathogens that cycle between humans and arthropods. This host alternation imposes a selective pressure on the viral population. ...Here, we found that dengue virus, the most important viral human pathogen transmitted by insects, evolved a mechanism to differentially regulate the production of viral non-coding RNAs in mosquitos and humans, with a significant impact on viral fitness in each host. Flavivirus infections accumulate non-coding RNAs derived from the viral 3'UTRs (known as sfRNAs), relevant in viral pathogenesis and immune evasion. We found that dengue virus host adaptation leads to the accumulation of different species of sfRNAs in vertebrate and invertebrate cells. This process does not depend on differences in the host machinery; but it was found to be dependent on the selection of specific mutations in the viral 3'UTR. Dissecting the viral population and studying phenotypes of cloned variants, the molecular determinants for the switch in the sfRNA pattern during host change were mapped to a single RNA structure. Point mutations selected in mosquito cells were sufficient to change the pattern of sfRNAs, induce higher type I interferon responses and reduce viral fitness in human cells, explaining the rapid clearance of certain viral variants after host change. In addition, using epidemic and pre-epidemic Zika viruses, similar patterns of sfRNAs were observed in mosquito and human infected cells, but they were different from those observed during dengue virus infections, indicating that distinct selective pressures act on the 3'UTR of these closely related viruses. In summary, we present a novel mechanism by which dengue virus evolved an RNA structure that is under strong selective pressure in the two hosts, as regulator of non-coding RNA accumulation and viral fitness. This work provides new ideas about the impact of host adaptation on the variability and evolution of flavivirus 3'UTRs with possible implications in virulence and viral transmission.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mosquitoes rely on RNA interference (RNAi) as their primary defense against viral infections. To this end, the combination of RNAi and invertebrate cell culture systems has become an invaluable tool ...in studying virus-vector interactions. Nevertheless, a recent study failed to detect an active RNAi response to West Nile virus (WNV) infection in C6/36 (Aedes albopictus) cells, a mosquito cell line frequently used to study arthropod-borne viruses (arboviruses). Therefore, we sought to determine if WNV actively evades the host's RNAi response or if C6/36 cells have a dysfunctional RNAi pathway. C6/36 and Drosophila melanogaster S2 cells were infected with WNV (Flaviviridae), Sindbis virus (SINV, Togaviridae) and La Crosse virus (LACV, Bunyaviridae) and total RNA recovered from cell lysates. Small RNA (sRNA) libraries were constructed and subjected to high-throughput sequencing. In S2 cells, virus-derived small interfering RNAs (viRNAs) from all three viruses were predominantly 21 nt in length, a hallmark of the RNAi pathway. However, in C6/36 cells, viRNAs were primarily 17 nt in length from WNV infected cells and 26-27 nt in length in SINV and LACV infected cells. Furthermore, the origin (positive or negative viral strand) and distribution (position along viral genome) of S2 cell generated viRNA populations was consistent with previously published studies, but the profile of sRNAs isolated from C6/36 cells was altered. In total, these results suggest that C6/36 cells lack a functional antiviral RNAi response. These findings are analogous to the type-I interferon deficiency described in Vero (African green monkey kidney) cells and suggest that C6/36 cells may fail to accurately model mosquito-arbovirus interactions at the molecular level.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract We established a laboratory colony of Culex pipiens mosquitoes from eggs collected in Colorado and discovered that mosquitoes in the colony are naturally infected with Culex flavivirus ...(CxFV), an insect-specific flavivirus. In this study we examined transmission dynamics of CxFV and effects of persistent CxFV infection on vector competence for West Nile virus (WNV). We found that vertical transmission is the primary mechanism for persistence of CxFV in Cx. pipiens , with venereal transmission potentially playing a minor role. Vector competence experiments indicated possible early suppression of WNV replication by persistent CxFV infection in Cx. pipiens . This is the first description of insect-specific flavivirus transmission dynamics in a naturally infected mosquito colony and the observation of delayed dissemination of superinfecting WNV suggests that the presence of CxFV may impact the intensity of enzootic transmission of WNV and the risk of human exposure to this important pathogen.
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