Sequence Homology and Tissue Tropism Determine Superinfection Exclusion of Zika virus in Aedes aegypti Mediated by an insect-specific Binjari-Zika Virus Chimera
It is demonstrated that incorporation of Zika virus prM and E proteins into the ISF backbone enhances infection of the mosquito midgut following oral exposure, which enables direct inhibition of ZIKV infection after a subsequent infectious blood meal, and defines a replication-dependent, tissue-specific mechanism of ISF-mediated protection.
Abstract
Arboviruses such as dengue, Zika, and chikungunya viruses cause widespread disease and continue to expand their geographical range due to climate change and vector spread. Insect-specific flaviviruses (ISFs) are promising biocontrol candidates of arboviruses, due to recent studies showing that prior infection with an ISF can reduce arbovirus replication in mosquitoes through superinfection exclusion (SIE). However, the route of infection, tissue tropism, pathogenesis and the mechanisms underlying SIE of ISFs in mosquitoes remain unclear. RNA interference (RNAi) is a potent antiviral response in insects, therefore it is expected that sequence homology between the ISF and the arbovirus will strengthen SIE. Here, we used ISF Binjari virus and a chimera containing the Zika virus structural proteins prME (BinJ-ZIKV) as a model system. Intrathoracic injection of BinJ-ZIKV in Aedes aegypti led to rapid systemic infection that excluded the midgut, subsequently blocking ZIKV dissemination from the midgut. SIE was strongest in tissues where primary-virus replication was highest. This spatial component of SIE was stronger when there was sequence homology between the ISF and arbovirus and displayed a strong 21nt siRNA response, suggesting RNAi contributed to the observed SIE. Upon oral inoculation, BinJ-ZIKV replicated efficiently in mosquitoes, was detected across multiple tissues, and saliva. BinJ-ZIKV also had higher infection establishment than BinJV at lower oral titres. SIE was observed for BinJ-ZIKV infection after oral exposure interfered with subsequent ZIKV midgut infection. Together, these findings support engineered ISF-chimeras as valuable experimental tools to dissect viral determinants of SIE and to optimize mosquito-based arbovirus interference strategies. Importance Annually, over 400 million people are infected with mosquito-transmitted viruses. Insect-specific flaviviruses (ISFs) can interfere with the transmission of clinically important viruses through a phenomenon termed superinfection exclusion (SIE). However, the mechanisms of SIE remain poorly understood. Using a Binjari virus chimera expressing Zika virus (ZIKV) structural proteins, we show that SIE is highly tissue-specific, with exclusion of ZIKV only occurring at sites where the chimera actively replicates and induces the mosquito antiviral RNA interference pathway. We further demonstrate that incorporation of Zika virus prM and E proteins into the ISF backbone enhances infection of the mosquito midgut following oral exposure, which enables direct inhibition of ZIKV infection after a subsequent infectious blood meal. Together, these findings define a replication-dependent, tissue-specific mechanism of ISF-mediated protection and provide a framework for reducing mosquito-borne virus transmission through SIE.
Globalization and climate change have contributed to the emergence and spread of arboviruses, with the possibility of their cocirculation in the same geographic region at the same time. Understanding the effects of concurrent infections within a mosquito vector or human host is therefore an emerging focus in arboviral research. Studies using mosquito- and mammalian-derived cell lines provide a controlled framework for studying viral replication dynamics. In this study, Vero cells were infected with either Zika virus (ZIKV) and West Nile virus (WNV) or ZIKV and Mayaro virus (MAYV). In nature, concurrent infections can occur either simultaneously (i.e., coinfections) or sequentially (i.e., superinfections), and both conditions were studied here. Arbovirus replication was assessed 6 days post-infection using RT-qPCR, along with corresponding cytopathic effects on cells. Single infections were used as comparators to the coinfection and superinfection assays. Coinfections (ZIKV/WNV and ZIKV/MAYV) resulted in viral interference, with ZIKV RNA levels reduced when cells were coinfected with either WNV or MAYV. Viral accommodation was observed when ZIKV was the first to infect the cells during superinfections. In contrast, superinfection exclusion was observed when ZIKV was introduced to cells that were already infected with another virus. RNA production of WNV and MAYV was not affected by the presence of ZIKV in any of the conditions investigated. Understanding replication dynamics in in vitro coinfection and superinfection scenarios is the first step toward addressing the knowledge gap concerning cocirculating arboviruses.
L. Krokovsky, Mariana Garrido de Castro, Amanda J. M. Williams et al.· PLoS ONE· 0 citations
ABSTRACT Mosquito cell lines are essential tools for arbovirus research. Endogenous viral elements (EVEs) are prevalent in mosquito genomes, yet their functional effects on host immune responses remain unclear, potentially complicating experimental interpretations. In this study, we systematically characterized endogenous mosquito densovirus-like elements (EMLs) within the Aedes aegypti Aag2 cell line and found that these endogenous EMLs are transcriptionally active but translationally defective. The silencing of EML transcripts significantly diminished the replication of Zika virus (ZIKV), Japanese encephalitis virus (JEV), and chikungunya virus (CHIKV), while transiently increasing dengue virus 2 (DENV-2), thereby indicating a virus-dependent regulatory mechanism. Mechanistically, RNA sequencing after EML interference, alongside plasmid-based mimic expression, demonstrated that EML transcripts downregulate defensin A, an antimicrobial peptide produced by mosquitoes. Functional assays using synthetic defensin A showed that this peptide differentially regulates arboviral infection. Binding assays and structural modeling further supported its interaction with viral envelope proteins, while stage-restricted infection assays revealed distinct stages of action: defensin A enhanced adsorption of ZIKV, JEV, and CHIKV, but did not promote DENV-2 adsorption or entry, and instead reduced DENV-2 RNA accumulation at the post-entry replication stage. Our findings highlight a previously unrecognized role of densovirus-derived EVEs in mosquito innate immunity, extending their functional scope from the well-established PIWI-interacting RNA-mediated antiviral defense to the regulation of antimicrobial peptide-associated immune pathways. These findings emphasize the necessity of accounting for EVE activity when analyzing data derived from mosquito cell lines, and suggest that related EVE-mediated immune regulation may contribute to arbovirus dynamics in mosquitoes. IMPORTANCE Mosquito-borne viruses such as dengue, Zika, Japanese encephalitis, and chikungunya continue to threaten human health worldwide. Laboratory studies often use Aedes aegypti cell lines to investigate how these viruses interact with their mosquito hosts. Here, we show that the genomes of these cells contain endogenous viral elements derived from mosquito densoviruses. Far from being inert fossils, these sequences are transcriptionally active and regulate mosquito immunity by suppressing the antimicrobial peptide defensin A. This immune modulation influences the replication of different arboviruses in opposite ways, enhancing some while restricting others. Our findings reveal that integrated viral elements can shape the outcome of arbovirus infection, with important implications for interpreting mosquito cell culture experiments and for evaluating endogenous viral element-mediated immune regulation in mosquito–virus interactions. Mosquito-borne viruses such as dengue, Zika, Japanese encephalitis, and chikungunya continue to threaten human health worldwide. Laboratory studies often use Aedes aegypti cell lines to investigate how these viruses interact with their mosquito hosts. Here, we show that the genomes of these cells contain endogenous viral elements derived from mosquito densoviruses. Far from being inert fossils, these sequences are transcriptionally active and regulate mosquito immunity by suppressing the antimicrobial peptide defensin A. This immune modulation influences the replication of different arboviruses in opposite ways, enhancing some while restricting others. Our findings reveal that integrated viral elements can shape the outcome of arbovirus infection, with important implications for interpreting mosquito cell culture experiments and for evaluating endogenous viral element-mediated immune regulation in mosquito–virus interactions.
Jie Xiao, Tian-Tian Wei, Hong-Zheng Lu et al.· Msphere· 0 citations
Mosquitoes transmit arboviruses that represent major global public health challenges. Increasing insecticide resistance and absence of effective antiviral therapies underscore the need for novel vector control strategies. Insect-specific viruses have emerged as candidates for biological control, however, the cellular mechanisms underlying their interactions with mosquito hosts remain poorly understood. Here, we examined the immune response of Aedes albopictus U4.4 cells to Kamiti River virus (KRV) infection, an insect-specific flavivirus. Cells were infected with KRV, and transcriptomic and small RNA profiles were analyzed at 24, 48 and 72 h post-infection. KRV infection induced production of virus-derived small interfering RNAs (vsiRNAs) and virus-derived PIWI-interacting RNA (vpiRNAs) from 24 to 72 h. The vsiRNAs predominantly mapped to the 3′ untranslated region of the KRV genome, whereas vpiRNAs formed distinct hotspots in regions encoding the NS1, NS3, NS4A/B and NS5 proteins. Transcriptomic analysis revealed upregulation of genes associated with the humoral immune response, including defensin, cecropin, and glutathione S-transferase, and downregulation of Toll-like receptors and ecdysone-induced transcripts at later stages of infection. These gene expression patterns suggest an early activation followed by suppression of key immune signaling pathways. Collectively, the findings indicate that KRV leads to coordinated modulation of antiviral RNAi and host transcriptional responses, consistent with a balanced, commensal-like interaction in mosquito cells.
Members of the Flaviviridae encode XRN1-resistant RNA structures (xrRNAs) within their 3′ untranslated regions (UTRs) that stall the cellular 5′→3′ exoribonuclease 1 (XRN1), resulting in the accumulation of subgenomic flavivirus RNAs (sfRNAs). Zika virus (ZIKV) contains two such elements, xrRNA1 and xrRNA2, which generate sfRNA1 and sfRNA2, respectively. A third sfRNA species (sfRNA3) has also been described. Building on evidence that sequences within the orthoflavivirus UTRs modulate host range, we replaced the xrRNA1 element in the ZIKV genome with xrRNA1 or xrRNA1-like elements from nine evolutionarily divergent flaviviruses and assessed the in vitro host ranges of the resulting chimeric viruses to determine whether xrRNA1 contributes to host specificity. We selected mosquito-borne, tick-borne, insect-specific, and vertebrate-specific orthoflaviviruses, together with representative viruses from the Pestivirus, Pegivirus, and Hepacivirus genera. All genetic exchanges yielded chimeric viruses that replicated in both vertebrate (Vero) and mosquito (C6/36) cells. Wild-type ZIKV replicated to substantially higher titers than all chimeric viruses in Vero cells but showed no consistent replication advantage in C6/36 cells. All chimeric viruses produced sfRNA1, whereas detection of sfRNA2 and sfRNA3 varied according to the substituted xrRNA1 sequence and host cell type, indicating that heterologous xrRNA1 substitution can alter downstream sfRNA production in a host cell-dependent manner. In summary, replacement of the ZIKV xrRNA1 element with evolutionarily divergent xrRNA1 or xrRNA1-like sequences did not alter in vitro host range or abolish sfRNA1 production, but substantially reduced viral fitness in vertebrate cells.
Chandra S. Tangudu, Wichan Dankaona, Nile L. Stoltz et al.· Viruses· 0 citations
Zika virus (ZIKV) is an arthropod-borne Orthoflavivirus that caused a major outbreak in Central and South America in 2015–16. During that outbreak, ZIKV infection during pregnancy was associated with high rates of adverse outcomes, including miscarriage and a spectrum of neurological birth defects collectively termed congenital Zika syndrome (CZS). Ten years later, there are still no licensed ZIKV vaccines, underscoring the need for robust experimental models to evaluate vaccine efficacy against congenital disease. Here, we evaluated a highly pathogenic nonhuman primate pregnancy model for its ability to assess vaccine-mediated protection using a Zika virus-like particle (VLP) vaccine as a test platform. First, non-pregnant rhesus macaques (RM) were vaccinated with a prime-boost method to compare control, VLP alone, and VLP plus Alhydrogel (alum) adjuvant. Vaccination plus alum elicited strong neutralizing antibody responses and reduced viral dissemination compared to controls. Next, we evaluated the vaccine’s protective efficacy against adverse pregnancy complications. Two cohorts of female RM were vaccinated with ZIKV-VLP adjuvanted with alum or alum alone prior to time-mated breeding. At gestational day (GD) 30 (mid first trimester), pregnant animals were challenged with ZIKV-DAK 41524, an African-lineage strain previously shown to induce frequent first-trimester pregnancy loss. Within the vaccinated cohort, two of three animals reached the study endpoint of GD 90 with no observed adverse pregnancy outcomes. One vaccinated animal experienced early pregnancy loss despite the absence of detectable virus in fetal or placental tissues. In the sham vaccine control pregnancy cohort, severe adverse outcomes included miscarriage and hydrops fetalis with widespread viral RNA and placental pathology in two animals. These results support a high risk of early pregnancy loss following African-lineage ZIKV-DAK 41524 infection in RM. This model can be further used to understand the complexities of placental immunological features underlying miscarriage following ZIKV infection.
Hannah K. Jaeger, Jessica L. Smith, C. Labriola et al.· PLoS Neglected Tropical Dise...· 0 citations
Alphaviruses are arthropod-borne viruses that recognize cellular receptors in both vertebrate hosts and mosquito vectors to complete their transmission cycle, yet how they maintain recognition of receptors across evolutionarily divergent host species remains unresolved. Among alphaviruses, chikungunya virus (CHIKV), which is primarily vectored in urban settings by Aedes species mosquitoes, is the most widespread, and causes explosive outbreaks that can involve hundreds of thousands to millions of cases annually. The cell adhesion protein Lachesin is a mosquito-specific cellular receptor for CHIKV and multiple other arthritogenic alphaviruses. The envelope E2–E1 glycoproteins of these alphaviruses broadly recognize Lachesin orthologs from diverse mosquito species, but not other insects or arachnids. Lachesin genetic manipulation to prevent mosquito virus infection without interfering with endogenous receptor function could have a major impact for CHIKV control. Here, we determined high-resolution cryo-electron microscopy (cryo-EM) structures of alphaviruses bound to Aedes albopictus Lachesin. Comparative analysis of Lachesin-bound CHIKV, Semliki Forest virus (SFV), and Middelburg virus (MIDV) revealed that these three genetically divergent viruses use a similar surface to recognize Lachesin domain 1, but with reorganized E2–E1 glycoprotein contact residues. We show that a soluble Ae. albopictus Lachesin receptor decoy protein blocks the E2–E1-mediated entry of CHIKV and other arthritogenic alphaviruses into mammalian cells with greater breadth than a vertebrate receptor MXRA8 decoy and protects against lethal SFV challenge and CHIKV pathogenesis in murine models. Additionally, we identified a naturally occurring single residue Lachesin polymorphism that is found in some Anopheles (malaria vector) mosquitoes, and fully ablates CHIKV E2–E1 recognition, informing strategies for mosquito-targeted genetic interventions that could prevent mosquito vector infection and virus transmission. These findings define distinct determinants of receptor binding in mosquitoes and humans for arthritogenic alphaviruses, with implications for countermeasure development and outbreak preparedness.
Xiao-Yi Fan, Wanyu Li, Jesse S. Plung et al.· bioRxiv· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
