RESUMEN
Mosquitoes are vectors for arboviruses, such as dengue, Zika, and Chikungunya. Symbiotic interactions can affect the intrinsic ability of mosquitoes to acquire and transmit arboviruses, referred to as vector competence. Insect-specific viruses (ISVs) are commonly found in symbiotic associations with mosquitoes in the wild and can affect many aspects of mosquito biology. Here, we review current knowledge on the effects of symbiotic ISV-mosquito interactions on vector competence. We discuss potential mechanisms underlying these interactions and their implications for shaping new biological control strategies. Finally, we highlight the need for field data analyzing the circulation of ISVs in mosquitoes associated with mechanistic studies in the laboratory.
Asunto(s)
Arbovirus , Mosquitos Vectores , Simbiosis , Animales , Mosquitos Vectores/virología , Mosquitos Vectores/fisiología , Arbovirus/fisiología , Virus de Insectos/fisiología , Culicidae/virología , Culicidae/fisiología , Infecciones por Arbovirus/transmisiónRESUMEN
BACKGROUND: Mosquito borne viruses, such as dengue, Zika, yellow fever and Chikungunya, cause millions of infections every year. These viruses are mostly transmitted by two urban-adapted mosquito species, Aedes aegypti and Aedes albopictus. Although mechanistic understanding remains largely unknown, Aedes mosquitoes may have unique adaptations that lower the impact of viral infection. Recently, we reported the identification of an Aedes specific double-stranded RNA binding protein (dsRBP), named Loqs2, that is involved in the control of infection by dengue and Zika viruses in mosquitoes. Preliminary analyses suggested that the loqs2 gene is a paralog of loquacious (loqs) and r2d2, two co-factors of the RNA interference (RNAi) pathway, a major antiviral mechanism in insects. RESULTS: Here we analyzed the origin and evolution of loqs2. Our data suggest that loqs2 originated from two independent duplications of the first double-stranded RNA binding domain of loqs that occurred before the origin of the Aedes Stegomyia subgenus, around 31 million years ago. We show that the loqs2 gene is evolving under relaxed purifying selection at a faster pace than loqs, with evidence of neofunctionalization driven by positive selection. Accordingly, we observed that Loqs2 is localized mainly in the nucleus, different from R2D2 and both isoforms of Loqs that are cytoplasmic. In contrast to r2d2 and loqs, loqs2 expression is stage- and tissue-specific, restricted mostly to reproductive tissues in adult Ae. aegypti and Ae. albopictus. Transgenic mosquitoes engineered to express loqs2 ubiquitously undergo developmental arrest at larval stages that correlates with massive dysregulation of gene expression without major effects on microRNAs or other endogenous small RNAs, classically associated with RNA interference. CONCLUSIONS: Our results uncover the peculiar origin and neofunctionalization of loqs2 driven by positive selection. This study shows an example of unique adaptations in Aedes mosquitoes that could ultimately help explain their effectiveness as virus vectors.
Asunto(s)
Aedes , Dengue , Infección por el Virus Zika , Virus Zika , Animales , Aedes/genética , Proteínas Portadoras/genética , Mosquitos Vectores/genética , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Virus Zika/genética , Virus Zika/metabolismoRESUMEN
The Asian bush mosquito Aedes japonicus is rapidly invading North America and Europe. Due to its potential to transmit multiple pathogenic arthropod-borne (arbo)viruses including Zika virus, West Nile virus, and chikungunya virus, it is important to understand the biology of this vector mosquito in more detail. In addition to arboviruses, mosquitoes can also carry insect-specific viruses that are receiving increasing attention due to their potential effects on host physiology and arbovirus transmission. In this study, we characterized the collection of viruses, referred to as the virome, circulating in Ae. japonicus populations in the Netherlands and France. Applying a small RNA-based metagenomic approach to Ae. japonicus, we uncovered a distinct group of viruses present in samples from both the Netherlands and France. These included one known virus, Ae. japonicus narnavirus 1 (AejapNV1), and three new virus species that we named Ae. japonicus totivirus 1 (AejapTV1), Ae. japonicus anphevirus 1 (AejapAV1) and Ae. japonicus bunyavirus 1 (AejapBV1). We also discovered sequences that were presumably derived from two additional novel viruses: Ae. japonicus bunyavirus 2 (AejapBV2) and Ae. japonicus rhabdovirus 1 (AejapRV1). All six viruses induced strong RNA interference responses, including the production of twenty-one nucleotide-sized small interfering RNAs, a signature of active replication in the host. Notably, AejapBV1 and AejapBV2 belong to different viral families; however, no RNA-dependent RNA polymerase sequence has been found for AejapBV2. Intriguingly, our small RNA-based approach identified an â¼1-kb long ambigrammatic RNA that is associated with AejapNV1 as a secondary segment but showed no similarity to any sequence in public databases. We confirmed the presence of AejapNV1 primary and secondary segments, AejapTV1, AejapAV1, and AejapBV1 by reverse transcriptase polymerase chain reaction (PCR) in wild-caught Ae. japonicus mosquitoes. AejapNV1 and AejapTV1 were found at high prevalence (87-100 per cent) in adult females, adult males, and larvae. Using a small RNA-based, sequence-independent metagenomic strategy, we uncovered a conserved and prevalent virome among Ae. japonicus mosquito populations. The high prevalence of AejapNV1 and AejapTV1 across all tested mosquito life stages suggests that these viruses are intimately associated with Ae. japonicus.
RESUMEN
Characterization of double-stranded (ds)RNAs is relevant to the understanding of viral replication and immune sensing. Here, we provide a protocol describing the use of anti-dsRNA antibodies for immunofluorescence and immunoblotting in virus-infected insect cells, which can also be applied to tissues and other organisms. We describe the procedures to prepare insect cells for viral infection, followed by RNA extraction and in vitro production of synthetic dsRNA controls. We then detail the steps for dsRNA detection by immunoblotting and immunofluorescence. For complete details on the use and execution of this protocol, please refer to de Faria et al. (2022).1.
Asunto(s)
Virus de Insectos , Insectos , ARN Bicatenario , Insectos/citología , Insectos/virología , Virus de Insectos/genética , Técnica del Anticuerpo Fluorescente , ImmunoblottingRESUMEN
Aedes aegypti and A. albopictus mosquitoes are the main vectors for dengue virus (DENV) and other arboviruses, including Zika virus (ZIKV). Understanding the factors that affect transmission of arboviruses from mosquitoes to humans is a priority because it could inform public health and targeted interventions. Reasoning that interactions among viruses in the vector insect might affect transmission, we analysed the viromes of 815 urban Aedes mosquitoes collected from 12 countries worldwide. Two mosquito-specific viruses, Phasi Charoen-like virus (PCLV) and Humaita Tubiacanga virus (HTV), were the most abundant in A. aegypti worldwide. Spatiotemporal analyses of virus circulation in an endemic urban area revealed a 200% increase in chances of having DENV in wild A. aegypti mosquitoes when both HTV and PCLV were present. Using a mouse model in the laboratory, we showed that the presence of HTV and PCLV increased the ability of mosquitoes to transmit DENV and ZIKV to a vertebrate host. By transcriptomic analysis, we found that in DENV-infected mosquitoes, HTV and PCLV block the downregulation of histone H4, which we identify as an important proviral host factor in vivo.
Asunto(s)
Aedes , Arbovirus , Virus del Dengue , Dengue , Virus de Insectos , Virus ARN , Infección por el Virus Zika , Virus Zika , Animales , Humanos , Virus Zika/genética , Virus de Insectos/fisiología , Virus del Dengue/genética , Mosquitos Vectores , Arbovirus/genéticaRESUMEN
Arboviruses (an acronym for "arthropod-borne virus"), such as dengue, yellow fever, Zika, and Chikungunya, are important human pathogens transmitted by mosquitoes. These viruses impose a growing burden on public health. Despite laboratory mice having been used for decades for understanding the basic biological phenomena of these viruses, it was only recently that researchers started to develop immunocompromised animals to study the pathogenesis of arboviruses and their transmission in a way that parallels natural cycles. Here, we show that the AG129 mouse (IFN α/ß/γ R-/-) is a suitable and comprehensive vertebrate model for studying the mosquito vector competence for the major arboviruses of medical importance, namely the dengue virus (DENV), yellow fever virus (YFV), Zika virus (ZIKV), Mayaro virus (MAYV), and Chikungunya virus (CHIKV). We found that, after intraperitoneal injection, AG129 mice developed a transient viremia lasting several days, peaking on day two or three post infection, for all five arboviruses tested in this study. Furthermore, we found that the observed viremia was ample enough to infect Aedes aegypti during a blood meal from the AG129 infected mice. Finally, we demonstrated that infected mosquitoes could transmit each of the tested arboviruses back to naïve AG129 mice, completing a full transmission cycle of these vector-borne viruses. Together, our data show that A129 mice are a simple and comprehensive vertebrate model for studies of vector competence, as well as investigations into other aspects of mosquito biology that can affect virus-host interactions.
RESUMEN
dsRNA sensing triggers antiviral responses against RNA and DNA viruses in diverse eukaryotes. In Drosophila, Invertebrate iridescent virus 6 (IIV-6), a large DNA virus, triggers production of small interfering RNAs (siRNAs) by the dsRNA sensor Dicer-2. Here, we show that host RNA polymerase II (RNAPII) bidirectionally transcribes specific AT-rich regions of the IIV-6 DNA genome to generate dsRNA. Both replicative and naked IIV-6 genomes trigger production of dsRNA in Drosophila cells, implying direct sensing of invading DNA. Loquacious-PD, a Dicer-2 co-factor essential for the biogenesis of endogenous siRNAs, is dispensable for processing of IIV-6-derived dsRNAs, which suggests that they are distinct. Consistent with this finding, inhibition of the RNAPII co-factor P-TEFb affects the synthesis of endogenous, but not virus-derived, dsRNA. Altogether, our results suggest that a non-canonical RNAPII complex recognizes invading viral DNA to synthesize virus-derived dsRNA, which activates the antiviral siRNA pathway in Drosophila.
Asunto(s)
ADN Viral , Drosophila , Animales , Antivirales , Virus ADN/genética , Drosophila/metabolismo , Iridovirus , Interferencia de ARN , ARN Polimerasa II/metabolismo , ARN Bicatenario/genética , ARN Interferente Pequeño/metabolismo , ARN Viral/metabolismoRESUMEN
Aedes aegypti mosquitoes are vectors for arboviruses of medical importance such as dengue (DENV) and Zika (ZIKV) viruses. Different innate immune pathways contribute to the control of arboviruses in the mosquito vector including RNA interference, Toll and Jak-STAT pathways. However, the role of cellular responses mediated by circulating macrophage-like cells known as hemocytes remains unclear. Here we show that hemocytes are recruited to the midgut of Ae. aegypti mosquitoes in response to DENV or ZIKV. Blockade of the phagocytic function of hemocytes using latex beads induced increased accumulation of hemocytes in the midgut and a reduction in virus infection levels in this organ. In contrast, inhibition of phagocytosis by hemocytes led to increased systemic dissemination and replication of DENV and ZIKV. Hence, our work reveals a dual role for hemocytes in Ae. aegypti mosquitoes, whereby phagocytosis is not required to control viral infection in the midgut but is essential to restrict systemic dissemination. Further understanding of the mechanism behind this duality could help the design of vector-based strategies to prevent transmission of arboviruses.
Asunto(s)
Aedes/citología , Aedes/virología , Virus del Dengue/fisiología , Hemocitos/inmunología , Hemocitos/virología , Virus Zika/fisiología , Aedes/anatomía & histología , Animales , Femenino , Hemocitos/fisiología , Mosquitos Vectores , Fagocitos/virología , FagocitosisRESUMEN
Mosquitoes are the major vectors for arthropod-borne viruses (arboviruses) of medical importance. Aedes aegypti and A. albopictus are the most prolific and widespread mosquito vectors being responsible for global transmission of dengue, Zika and Chikungunya viruses. Characterizing the collection of viruses circulating in mosquitoes, the virome, has long been of special interest. In addition to arboviruses, mosquitoes carry insect-specific viruses (ISVs) that do not directly infect vertebrates. Mounting evidence indicates that ISVs interact with arboviruses and may affect mosquito vector competence. Here, we review our current knowledge about the virome of vector mosquitoes and discuss the challenges for the field that may lead to novel strategies to prevent outbreaks of arboviruses.
Asunto(s)
Arbovirus/fisiología , Culicidae/virología , Virus de Insectos/fisiología , Mosquitos Vectores/virología , Viroma , Animales , Infecciones por Arbovirus/transmisión , Infecciones por Arbovirus/virología , Arbovirus/clasificación , Interacciones Microbiota-Huesped , Humanos , Virus de Insectos/clasificación , Interacciones Microbianas , FilogeniaRESUMEN
The emergence of new human viral pathogens and re-emergence of several diseases are of particular concern in the last decades. Oropouche orthobunyavirus (OROV) is an arbovirus endemic to South and Central America tropical regions, responsible to several epidemic events in the last decades. There is little information regarding the ability of OROV to be transmitted by urban/peri-urban mosquitoes, which has limited the predictability of the emergence of permanent urban transmission cycles. Here, we evaluated the ability of OROV to infect, replicate, and be transmitted by three anthropophilic and urban species of mosquitoes, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus. We show that OROV is able to infect and efficiently replicate when systemically injected in all three species tested, but not when orally ingested. Moreover, we find that, once OROV replication has occurred in the mosquito body, all three species were able to transmit the virus to immunocompromised mice during blood feeding. These data provide evidence that OROV is restricted by the midgut barrier of three major urban mosquito species, but, if this restriction is overcome, could be efficiently transmitted to vertebrate hosts. This poses a great risk for the emergence of permanent urban cycles and geographic expansion of OROV to other continents.
Asunto(s)
Aedes/virología , Culex/virología , Mosquitos Vectores/virología , Orthobunyavirus/fisiología , Animales , Infecciones por Bunyaviridae/transmisión , Infecciones por Bunyaviridae/virología , Modelos Animales de Enfermedad , Femenino , Especificidad del Huésped , Interacciones Huésped-Patógeno , Ratones , Ratones NoqueadosRESUMEN
Huntington's disease (HD) is a genetic disorder marked by transcriptional alterations that result in neuronal impairment and death. MicroRNAs (miRNAs) are non-coding RNAs involved in post-transcriptional regulation and fine-tuning of gene expression. Several studies identified altered miRNA expression in HD and other neurodegenerative diseases, however their roles in early stages of HD remain elusive. Here, we deep-sequenced miRNAs from the striatum of the HD mouse model, BACHD, at the age of 2 and 8 months, representing the pre-symptomatic and symptomatic stages of the disease. Our results show that 44 and 26 miRNAs were differentially expressed in 2- and 8-month-old BACHD mice, respectively, as compared to wild-type controls. Over-representation analysis suggested that miRNAs up-regulated in 2-month-old mice control the expression of genes crucial for PI3K-Akt and mTOR cell signaling pathways. Conversely, miRNAs regulating genes involved in neuronal disorders were down-regulated in 2-month-old BACHD mice. Interestingly, primary striatal neurons treated with anti-miRs targeting two up-regulated miRNAs, miR-449c-5p and miR-146b-5p, showed higher levels of cell death. Therefore, our results suggest that the miRNAs altered in 2-month-old BACHD mice regulate genes involved in the promotion of cell survival. Notably, over-representation suggested that targets of differentially expressed miRNAs at the age of 8 months were not significantly enriched for the same pathways. Together, our data shed light on the role of miRNAs in the initial stages of HD, suggesting a neuroprotective role as an attempt to maintain or reestablish cellular homeostasis.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Enfermedad de Huntington/genética , MicroARNs/biosíntesis , MicroARNs/genética , Neuroprotección/fisiología , Síntomas Prodrómicos , Animales , Células Cultivadas , Femenino , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de Secuencia de ARN/métodos , Regulación hacia Arriba/fisiologíaRESUMEN
We previously reported that an ortholog of STING regulates infection by picorna-like viruses in Drosophila In mammals, STING is activated by the cyclic dinucleotide 2'3'-cGAMP produced by cGAS, which acts as a receptor for cytosolic DNA. Here, we showed that injection of flies with 2'3'-cGAMP induced the expression of dSTING-regulated genes. Coinjection of 2'3'-cGAMP with a panel of RNA or DNA viruses resulted in substantially reduced viral replication. This 2'3'-cGAMP-mediated protection was still observed in flies with mutations in Atg7 and AGO2, genes that encode key components of the autophagy and small interfering RNA pathways, respectively. By contrast, this protection was abrogated in flies with mutations in the gene encoding the NF-κB transcription factor Relish. Transcriptomic analysis of 2'3'-cGAMP-injected flies revealed a complex response pattern in which genes were rapidly induced, induced after a delay, or induced in a sustained manner. Our results reveal that dSTING regulates an NF-κB-dependent antiviral program that predates the emergence of interferons in vertebrates.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , Nucleótidos Cíclicos/metabolismo , Factores de Transcripción/metabolismo , Virus/metabolismo , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas de la Membrana/genética , Mutación , FN-kappa B/genética , Nucleótidos Cíclicos/genética , Factores de Transcripción/genética , Virus/genéticaRESUMEN
In vitro, Drosophila melanogaster Dicer-2 (Dcr-2) uses its helicase domain to initiate processing of dsRNA with blunt (BLT) termini, and its Platformâ¢PAZ domain to initiate processing of dsRNA with 3' overhangs (ovrs). To understand the relationship of these in vitro observations to roles of Dcr-2 in vivo, we compared in vitro effects of two helicase mutations to their impact on production of endogenous and viral siRNAs in flies. Consistent with the importance of the helicase domain in processing BLT dsRNA, both point mutations eliminated processing of BLT, but not 3'ovr, dsRNA in vitro. However, the mutations had different effects in vivo. A point mutation in the Walker A motif of the Hel1 subdomain, G31R, largely eliminated production of siRNAs in vivo, while F225G, located in the Hel2 subdomain, showed reduced levels of endogenous siRNAs, but did not significantly affect virus-derived siRNAs. In vitro assays monitoring dsRNA cleavage, dsRNA binding, ATP hydrolysis, and binding of the accessory factor Loquacious-PD provided insight into the different effects of the mutations on processing of different sources of dsRNA in flies. Our in vitro studies suggest effects of the mutations in vivo relate to their effects on ATPase activity, dsRNA binding, and interactions with Loquacious-PD. Our studies emphasize the importance of future studies to characterize dsRNA termini as they exist in Drosophila and other animals.
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Adenosina Trifosfato/metabolismo , ADN Helicasas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Mutación , ARN Helicasas/metabolismo , ARN Bicatenario/metabolismo , Ribonucleasa III/metabolismo , Animales , ADN Helicasas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimología , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Técnicas In Vitro , Masculino , MicroARNs/genética , ARN Helicasas/genética , ARN Bicatenario/genética , ARN Interferente Pequeño/genética , Ribonucleasa III/genéticaRESUMEN
Mayaro virus (MAYV), a sylvatic arbovirus belonging to the Togaviridae family and Alphavirus genus, is responsible for an increasing number of outbreaks in several countries of Central and South America. Despite Haemagogus janthinomys being identified as the main vector of MAYV, laboratory studies have already demonstrated the competence of Aedes aegypti to transmit MAYV. It has also been demonstrated that the WolbachiawMel strain is able to impair the replication and transmission of MAYV in Ae. aegypti. In Ae. aegypti, the small interfering RNA (siRNA) pathway is an important antiviral mechanism; however, it remains unclear whether siRNA pathway acts against MAYV infection in Ae. aegypti. The main objective of this study was to determine the contribution of the siRNA pathway in the control of MAYV infection. Thus, we silenced the expression of AGO2, an essential component of the siRNA pathway, by injecting dsRNA-targeting AGO2 (dsAGO2). Our results showed that AGO2 is required to control MAYV replication upon oral infection in Wolbachia-free Ae. aegypti. On the other hand, we found that Wolbachia-induced resistance to MAYV in Ae. aegypti is independent of the siRNA pathway. Our study brought new information regarding the mechanism of viral protection, as well as on Wolbachia mediated interference.
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Aedes/microbiología , Aedes/virología , Alphavirus/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Wolbachia/fisiología , Aedes/inmunología , Infecciones por Alphavirus/inmunología , Infecciones por Alphavirus/virología , Animales , Femenino , Humanos , Inmunidad Innata , Mosquitos Vectores/inmunología , Mosquitos Vectores/microbiología , Mosquitos Vectores/virología , Wolbachia/inmunologíaRESUMEN
Insects are the most diverse group of animals. They can be infected by an extraordinary diversity of viruses. Among them, arthropod-borne viruses (arboviruses) can be transmitted to humans. High-throughput sequencing of small RNAs from insects provides insight into their virome, which may help understand the dynamics of vector borne infectious diseases. Furthermore, investigating the mechanisms that restrict viral infections in insects points to genetic innovations that may inspire novel antiviral strategies.
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Biodiversidad , Genoma Viral/genética , Insectos Vectores/virología , Virus de Insectos/clasificación , Virus ARN/clasificación , Animales , Insectos Vectores/clasificación , Virus de Insectos/genética , Virus ARN/genéticaRESUMEN
To uncover the factors that dictate the persistence of a memory, it is critical to determine the molecular basis of consolidation. Here, we submitted male adult C57/BL6 mice to contextual fear conditioning using 1US (US: foot-shock, 0.7 mA, 2 s) or 5US, to generate recent (24 to 48 h) and remote (30 days) memories, respectively. To access the functional role of de novo transcription, we injected actinomycin D (ActD: 2.5 ng/side) directly into the dorsal hippocampus (HIP) or dorsomedial prefrontal cortex (dmPFC), 0 (early consolidation) or 12 h (late consolidation) after training. Our results showed that de novo transcription at 0 h was required for recent and remote memories. However, 12 h was a critical time point to memory persistence. In the dHIP, de novo transcription at 12 h post-training differentiated the recent memory from the remote. In the dmPFC, ActD affected memory formation depending on the training intensity (1 or 5US). Specifically, freezing was amplified after 5US conditioning. Furthermore, inhibiting de novo transcription at 12 h post-training in the dmPFC rapidly increased c-Fos expression in the amygdala. Altogether, our results indicate that contextual fear memory duration is particularly sensitive to de novo transcription in the dHIP and dmPFC, at a specific time point of late consolidation.
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Miedo/fisiología , Hipocampo/fisiología , Consolidación de la Memoria/fisiología , Corteza Prefrontal/fisiología , Transcripción Genética , Amígdala del Cerebelo/fisiología , Animales , Biomarcadores/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Electrochoque , Regulación de la Expresión Génica , Masculino , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , ARN/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Arthropod vectors control the replication of arboviruses through their innate antiviral immune responses. In particular, the RNA interference (RNAi) pathways are of notable significance for the control of viral infections. Although much has been done to understand the role of RNAi in vector populations, little is known about its importance in non-vector mosquito species. In this study, we investigated the presence of an RNAi response in Toxorhynchites amboinensis, which is a non-blood feeding species proposed as a biological control agent against pest mosquitoes. Using a derived cell line (TRA-171), we demonstrate that these mosquitoes possess a functional RNAi response that is active against a mosquito-borne alphavirus, Semliki Forest virus. As observed in vector mosquito species, small RNAs are produced that target viral sequences. The size and characteristics of these small RNAs indicate that both the siRNA and piRNA pathways are induced in response to infection. Taken together, this data suggests that Tx. amboinensis are able to control viral infections in a similar way to natural arbovirus vector mosquito species. Understanding their ability to manage arboviral infections will be advantageous when assessing these and similar species as biological control agents.
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Culicidae/genética , Culicidae/virología , Interferencia de ARN , Virus de los Bosques Semliki/genética , Infecciones por Alphavirus/inmunología , Animales , Agentes de Control Biológico , Línea Celular , Culicidae/citología , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunidad Innata , Mosquitos Vectores/genética , Mosquitos Vectores/virología , ARN Interferente Pequeño/genética , Virus de los Bosques Semliki/inmunología , Replicación ViralRESUMEN
BACKGROUND: Hevea brasiliensis is an important commercial crop due to the high quality of the latex it produces; however, little is known about viral infections in this plant. The only virus described to infect H. brasiliensis until now is a Carlavirus, which was described more than 30 years ago. Virus-derived small interfering RNA (vsiRNAs) are the product of the plant's antiviral defense triggered by dsRNA viral intermediates generated, during the replication cycle. These vsiRNAs are complementar to viral genomes and have been widely used to identify and characterize viruses in plants. METHODS: In the present study, we investigated the virome of leaf and sapwood samples from native H. brasiliensis trees collected in two geographic areas in the Brazilian Amazon. Small RNA (sRNA) deep sequencing and bioinformatic tools were used to assembly, identify and characterize viral contigs. Subsequently, PCR amplification techniques were performed to experimentally verify the presence of the viral sequences. Finally, the phylogenetic relationship of the putative new virus with related viral genomes was analyzed. RESULTS: Our strategy allowed the identification of 32 contigs with high similarity to viral reference genomes, from which 23 exhibited homology to viruses of the Tymoviridae family. The reads showed a predominant size distribution at 21 nt derived from both strands, which was consistent with the vsiRNAs profile. The presence and genome position of the viral contigs were experimentally confirmed using droplet digital PCR amplifications. A 1913 aa long fragment was obtained and used to infer the phylogenetic relationship of the putative new virus, which indicated that it is taxonomically related to the Grapevine fleck virus, genus Maculavirus. The putative new virus was named Hevea brasiliensis virus (HBrV) in reference to its host. CONCLUSION: The methodological strategy applied here proved to be efficient in detecting and confirming the presence of new viral sequences on a 'very difficult to manage' sample. This is the second time that viral sequences, that could be ascribed as a putative novel virus, associated to the rubber tree has been identified.
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Hevea/virología , Virus ARN/clasificación , Virus ARN/aislamiento & purificación , ARN Interferente Pequeño/genética , Perfilación de la Expresión Génica , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Filogenia , Enfermedades de las Plantas/virología , Hojas de la Planta/virología , Reacción en Cadena de la Polimerasa , ARN Viral/genética , Análisis de Secuencia de ARNRESUMEN
Dengue virus (DENV) is an arbovirus transmitted to humans by Aedes mosquitoes1. In the insect vector, the small interfering RNA (siRNA) pathway is an important antiviral mechanism against DENV2-5. However, it remains unclear when and where the siRNA pathway acts during the virus cycle. Here, we show that the siRNA pathway fails to efficiently silence DENV in the midgut of Aedes aegypti although it is essential to restrict systemic replication. Accumulation of DENV-derived siRNAs in the midgut reveals that impaired silencing results from a defect downstream of small RNA biogenesis. Notably, silencing triggered by endogenous and exogenous dsRNAs remained effective in the midgut where known components of the siRNA pathway, including the double-stranded RNA (dsRNA)-binding proteins Loquacious and r2d2, had normal expression levels. We identified an Aedes-specific paralogue of loquacious and r2d2, hereafter named loqs2, which is not expressed in the midgut. Loqs2 interacts with Loquacious and r2d2 and is required to control systemic replication of DENV and also Zika virus. Furthermore, ectopic expression of Loqs2 in the midgut of transgenic mosquitoes is sufficient to restrict DENV replication and dissemination. Together, our data reveal a mechanism of tissue-specific regulation of the mosquito siRNA pathway controlled by Loqs2.
Asunto(s)
Aedes/metabolismo , Proteínas Portadoras/metabolismo , Virus del Dengue/metabolismo , Expresión Génica Ectópica , ARN Bicatenario/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Aedes/genética , Aedes/virología , Animales , Animales Modificados Genéticamente , Antivirales/metabolismo , Antivirales/farmacología , Proteínas Portadoras/genética , Replicación del ADN , Dengue/virología , Virus del Dengue/efectos de los fármacos , Virus del Dengue/genética , Virus del Dengue/patogenicidad , Proteínas de Drosophila , Femenino , Tracto Gastrointestinal/virología , Silenciador del Gen , Interacciones Huésped-Patógeno , Mosquitos Vectores/virología , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/farmacología , Replicación Viral , Virus Zika/metabolismoRESUMEN
Brucella abortus is a Gram-negative intracellular bacterium that causes a worldwide zoonosis termed brucellosis, which is characterized as a debilitating infection with serious clinical manifestations leading to severe complications. In spite of great advances in studies involving host-B. abortus interactions, there are many gaps related to B. abortus modulation of the host immune response through regulatory mechanisms. Here, we deep sequenced small RNAs from bone marrow-derived macrophages infected with B. abortus, identifying 69 microRNAs (miRNAs) that were differentially expressed during infection. We further validated the expression of four upregulated and five downregulated miRNAs during infection in vitro that displayed the same profile in spleens from infected mice at 1, 3, or 6 days post-infection. Among these miRNAs, mmu-miR-181a-5p (upregulated) or mmu-miR-21a-5p (downregulated) were selected for further analysis. First, we determined that changes in the expression of both miRNAs induced by infection were dependent on the adaptor molecule MyD88. Furthermore, evaluating putative targets of mmu-miR-181a-5p, we demonstrated this miRNA negatively regulates TNF-α expression following Brucella infection. By contrast, miR-21a-5p targets included a negative regulator of IL-10, programmed cell death protein 4, and several guanylate-binding proteins (GBPs). As a result, during infection, miR-21a-5p led to upregulation of IL-10 expression and downregulation of GBP5 in macrophages infected with Brucella. Since GBP5 and IL-10 are important molecules involved in host control of Brucella infection, we decided to investigate the role of mmu-miR-21a-5p in bacterial replication in macrophages. We observed that treating macrophages with a mmu-miR-21a-5p mimic enhanced bacterial growth, whereas transfection of its inhibitor reduced Brucella load in macrophages. Taken together, the results indicate that downregulation of mmu-miR-21a-5p induced by infection increases GBP5 levels and decreases IL-10 expression thus contributing to bacterial control in host cells.