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West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) are closely related flaviviruses that can cause encephalitis in humans and related diseases in animals. In nature, both are transmitted by Culex, with wild birds, including jays, sparrows, and robins, serving as vertebrate hosts. WNV and SLEV circulate in the same environments and have recently caused concurrent disease outbreaks in humans. The extent that coinfection of mosquitoes or birds may alter transmission dynamics, however, is not well characterized. We therefore sought to determine if coinfection alters infection kinetics and virus levels in birds and infection rates in mosquitoes. Accordingly, American robins (Turdus migratorius), two species of mosquitoes, and vertebrate and invertebrate cells were infected with WNV and/or SLEV to assess how simultaneous exposure may alter infection outcomes. There was variable impact of coinfection in vertebrate cells, with some evidence that SLEV can suppress WNV replication. However, robins had comparable viremia and antibody responses regardless of coinfection. Conversely, in Culex cells and mosquitoes, we saw a minimal impact of simultaneous exposure to both viruses on replication, with comparable infection, dissemination, and transmission rates in singly infected and coinfected mosquitoes. Importantly, while WNV and SLEV levels in coinfected mosquito midguts were positively correlated, we saw no correlation between them in salivary glands and saliva. These results reveal that while coinfection can occur in both avian and mosquito hosts, the viruses minimally impact one another. The potential for coinfection to alter virus population structure or the likelihood of rare genotypes emerging remains unknown. [ABSTRACT FROM AUTHOR]

Systemic viral infection of insects typically begins with the primary infection of midgut epithelial cells (enterocytes) and subsequent transit of the progeny virus in an apical-to-basal orientation into the hemocoel. For insect-vectored viruses, an oppositely oriented process (basal-to-apical transit) occurs upon secondary infection of salivary glands and is necessary for virus transmission to non-insect hosts. To examine this inversely oriented virus transit in these polarized tissues, we assessed the intracellular trafficking of two model viral envelope proteins (baculovirus GP64 and vesicular stomatitis virus G) in the midgut and salivary gland cells of the model insect, Drosophila melanogaster. Using fly lines that inducibly express either GP64 or VSV G, we found that each protein, expressed alone, was trafficked basally in midgut enterocytes. In salivary gland cells, VSV G was trafficked apically in most but not all cells, whereas GP64 was consistently trafficked basally. We demonstrated that a YxxØ motif present in both proteins was critical for basal trafficking in midgut enterocytes but dispensable for trafficking in salivary gland cells. Using RNAi, we found that clathrin adaptor protein complexes AP-1 and AP-3, as well as seven Rab GTPases, were involved in polarized VSV G trafficking in midgut enterocytes. Our results indicate that these viral envelope proteins encode the requisite information and require no other viral factors for appropriately polarized trafficking. In addition, they exploit tissue-specific differences in protein trafficking pathways to facilitate virus egress in the appropriate orientation for establishing systemic infections and vectoring infection to other hosts. IMPORTANCE Viruses that use insects as hosts must navigate specific routes through different insect tissues to complete their life cycles. The routes may differ substantially depending on the life cycle of the virus. Both insect pathogenic viruses and insect-vectored viruses must navigate through the polarized cells of the midgut epithelium to establish a systemic infection. In addition, insect-vectored viruses must also navigate through the polarized salivary gland epithelium for transmission. Thus, insect-vectored viruses appear to traffic in opposite directions in these two tissues. In this study, we asked whether two viral envelope proteins (VSV G and baculovirus GP64) alone encode the signals necessary for the polarized trafficking associated with their respective life cycles. Using Drosophila as a model to examine tissue-specific polarized trafficking of these viral envelope proteins, we identified one of the virus-encoded signals and several host proteins associated with regulating the polarized trafficking in the midgut epithelium. [ABSTRACT FROM AUTHOR]

ABSTRACT As climate change alters Earth’s biomes, it is expected the transmission dynamics of mosquito-borne viruses will change. While the effects of temperature changes on mosquito-virus interactions and the spread of the pathogens have been elucidated over the last decade, the impact of relative humidity changes is still relatively unknown. To overcome this knowledge gap, we exposed Aedes aegypti females to various humidity conditions. We measured different components of vectorial capacity such as survival, blood-feeding rates, and changes in infection and dissemination of Zika virus. Survival decreased as the humidity level decreased, while infection rates increased as the humidity level decreased. Alternatively, blood feeding rates and disseminated infection rates peaked at the intermediate 50% relative humidity treatment but were the same in the 30% and 80% relative humidity treatments. These results provide empirical evidence that Ae. aegypti exposure to low humidity can enhance Zika virus infection in the mosquito, which has important implications in predicting how climate change will impact mosquito-borne viruses.IMPORTANCEViruses transmitted by mosquitoes to humans are a major public health burden and are expected to increase under climate change. While we know that temperature is an important driver of variation in arbovirus replication in the mosquito, very little is known about how other relevant climate variables such as humidity will influence the interaction between mosquitoes and the viruses they transmit. Given the variability in humidity across environments, and the predicted changes in humidity under climate change, it is imperative that we also study the impact that it has on mosquito infection and transmission of arboviruses.

ABSTRACTSandfly-borne phleboviruses (SBPs), which cause sandfly fever, aseptic meningitis, encephalitis, and meningoencephalitis, are emerging pathogens of major public health concern. Virus nucleic acid testing is essential for SBP diagnosis, especially in the early stages of infection, and for the discovery of novel SBPs. The efficacy of utilizing generic primers that target conserved nucleotide sequences for the detection of both known and novel SBPs has not been extensively evaluated. We aimed to compare and evaluate the performance of five generic primer sets, widely used to detect S- and L-segments of arthropod-borne phleboviruses and designed as singleplex (n = 3) and nested (n = 2) formats, including both well-known and recently characterized 15 Old World virus strains. Furthermore, we performed in silico analysis to assess the detection capabilities of these generic primer sets. The initial evaluation of previously published generic primer sets for SBP detection yielded two singleplex primer sets with the potential to be adapted for use in real-time or high-throughput detection settings. Studies are ongoing to develop and further optimize a preliminary assay and test various hosts and vectors to assess their capacity to detect known and novel viruses.IMPORTANCEVirus nucleic acid testing is the primary diagnostic method, particularly in the early stages of illness. Virus-specific or syndromic tests are widely used for this purpose. The use of generic primers has had a considerable impact on the discovery, identification, and detection of Old World sandfly-borne phleboviruses (OWSBP). The study is significant because it is the first to carry out a comparative evaluation of all published OWSBP generic primer sets.

ABSTRACT Wolbachia are a genus of insect endosymbiotic bacteria which includes strains wMel and wAlbB that are being utilized as a biocontrol tool to reduce the incidence of Aedes aegypti-transmitted viral diseases like dengue. However, the precise mechanisms underpinning the antiviral activity of these Wolbachia strains are not well defined. Here, we generated a panel of Ae. aegypti-derived cell lines infected with antiviral strains wMel and wAlbB or the non-antiviral Wolbachia strain wPip to understand host cell morphological changes specifically induced by antiviral strains. Antiviral strains were frequently found to be entirely wrapped by the host endoplasmic reticulum (ER) membrane, while wPip bacteria clustered separately in the host cell cytoplasm. ER-derived lipid droplets (LDs) increased in volume in wMel- and wAlbB-infected cell lines and mosquito tissues compared to cells infected with wPip or Wolbachia-free controls. Inhibition of fatty acid synthase (required for triacylglycerol biosynthesis) reduced LD formation and significantly restored ER-associated dengue virus replication in cells occupied by wMel. Together, this suggests that antiviral Wolbachia strains may specifically alter the lipid composition of the ER to preclude the establishment of dengue virus (DENV) replication complexes. Defining Wolbachia’s antiviral mechanisms will support the application and longevity of this effective biocontrol tool that is already being used at scale.IMPORTANCEAedes aegypti transmits a range of important human pathogenic viruses like dengue. However, infection of Ae. aegypti with the insect endosymbiotic bacterium, Wolbachia, reduces the risk of mosquito to human viral transmission. Wolbachia is being utilized at field sites across more than 13 countries to reduce the incidence of viruses like dengue, but it is not well understood how Wolbachia induces its antiviral effects. To examine this at the subcellular level, we compared how different strains of Wolbachia with varying antiviral strengths associate with and modify host cell structures. Strongly antiviral strains were found to specifically associate with the host endoplasmic reticulum and induce striking impacts on host cell lipid droplets. Inhibiting Wolbachia-induced lipid redistribution partially restored dengue virus replication demonstrating this is a contributing role for Wolbachia's antiviral activity. These findings provide new insights into how antiviral Wolbachia strains associate with and modify Ae. aegypti host cells.

ABSTRACTDespite numerous studies highlighting the higher transmissibility of the African Zika virus (ZIKV) lineage compared to the Asian lineage in mosquito vectors, little is known about how the viruses interact with different tissues during the early steps of mosquito infection. To address this gap, we aimed to characterize intra-host infection barriers by combining tissue-level monitoring of infection using plaque assays and a novel quantitative analysis of single-cell-level infection kinetics by in situ immunofluorescent staining. Our results revealed that, in Aedes aegypti, an African ZIKV strain exhibited a higher replication rate across various tissues than an Asian ZIKV strain. This difference was potentially due to a higher virus production in individual cells, faster spread within tissues, or a combination of both factors. Furthermore, we observed that higher blood meal titers resulted in a faster viral spread to neighboring cells suggesting that intra-host infection dynamics depend on inoculum size. We also identified a significant bottleneck during midgut infection establishment for both ZIKV lineages, with only a small percentage of the virus population successfully initiating infection. Finally, the in situ immunofluorescent staining technique enabled the examination of virus infection characteristics in different cell types and revealed heterogeneity in viral replication. Together, these findings demonstrate that differences in intra-host infection kinetics underlie differential transmissibility between African and Asian ZIKV lineages. This information could serve as a starting point to further investigate the underlying mechanisms and ultimately inform the development of alternative control strategies.IMPORTANCEThe recent Zika virus (ZIKV) epidemic in the Americas highlights its potential public health threat. While the Asian ZIKV lineage has been identified as the main cause of the epidemic, the African lineage, which has been primarily confined to Africa, has shown evidence of higher transmissibility in Aedes mosquitoes. To gain a deeper understanding of this differential transmissibility, our study employed a combination of tissue-level infection kinetics and single-cell-level infection kinetics using in situ immunofluorescent staining. We discovered that the African ZIKV lineage propagates more rapidly and spreads more efficiently within mosquito cells and tissues than its Asian counterpart. This information lays the groundwork for future exploration of the viral and host determinants driving these variations in propagation efficiency.

Zika virus (ZIKV) is now in a post-pandemic period, for which the potential for re-emergence and future spread is unknown. Adding to this uncertainty is the unique capacity of ZIKV to directly transmit between humans via sexual transmission. Recently, we demonstrated that direct transmission of ZIKV between vertebrate hosts leads to rapid adaptation resulting in enhanced virulence in mice and the emergence of three amino acid substitutions (NS2A-A117V, NS2A-A117T, and NS4A-E19G) shared among all vertebrate-passaged lineages. Here, we further characterized these host-adapted viruses and found that vertebrate-passaged viruses do not lose fitness or transmission potential in mosquitoes. To understand the contribution of genetic changes to the enhanced virulence and transmission phenotype, we engineered these amino acid substitutions, singly and in combination, into a ZIKV infectious clone. We found that NS4A-E19G contributed to the enhanced virulence and mortality phenotype in mice. Further analyses revealed that NS4A-E19G results in increased viral loads and distinct transcriptional patterns for innate immune genes in the brain. None of the substitutions contributed to changes in mosquito vector competence. Together, these findings suggest that direct transmission chains could enable the emergence of more virulent ZIKV strains without compromising mosquito transmission capacity, although the underlying genetics of these adaptations are complex. IMPORTANCE Previously, we modeled direct transmission chains of Zika virus (ZIKV) by serially passaging ZIKV in mice and mosquitoes and found that direct mouse transmission chains selected for viruses with increased virulence in mice and the acquisition of non-synonymous amino acid substitutions. Here, we show that these same mouse-passaged viruses also maintain fitness and transmission capacity in mosquitoes. We used infectious clone-derived viruses to demonstrate that the substitution in nonstructural protein 4A contributes to increased virulence in mice. [ABSTRACT FROM AUTHOR]

ABSTRACT Aedes aegypti mosquitoes can transmit several arboviruses, including chikungunya virus (CHIKV), dengue virus (DENV), and Zika virus (ZIKV). When blood-feeding on a virus-infected human, the mosquito ingests the virus into the midgut (stomach), where it replicates and must overcome the midgut barrier to disseminate to other organs and ultimately be transmitted via the saliva. Current tools to study mosquito-borne viruses (MBVs) include 2D-cell culture systems and in vivo mosquito infection models, which offer great advantages, yet have some limitations. Here, we describe a long-term ex vivo culture of Ae. aegypti guts. Cultured guts were metabolically active for 7 d in a 96-well plate at 28°C and were permissive to ZIKV, DENV, Ross River virus, and CHIKV. Ex vivo guts from Culex pipiens mosquitoes were found to be permissive to Usutu virus. Immunofluorescence staining confirmed viral protein synthesis in CHIKV-infected guts of Ae. aegypti. Furthermore, fluorescence microscopy revealed replication and spread of a reporter DENV in specific regions of the midgut. In addition, two known antiviral molecules, β-d-N4-hydroxycytidine and 7-deaza-2′-C-methyladenosine, were able to inhibit CHIKV and ZIKV replication, respectively, in the ex vivo model. Together, our results show that ex vivo guts can be efficiently infected with mosquito-borne alpha- and flaviviruses and employed to evaluate antiviral drugs. Furthermore, the setup can be extended to other mosquito species. Ex vivo gut cultures could thus be a new model to study MBVs, offering the advantage of reduced biosafety measures compared to infecting living mosquitoes. IMPORTANCE Mosquito-borne viruses (MBVs) are a significant global health threat since they can cause severe diseases in humans, such as hemorrhagic fever, encephalitis, and chronic arthritis. MBVs rely on the mosquito vector to infect new hosts and perpetuate virus transmission. No therapeutics are currently available. The study of arbovirus infection in the mosquito vector can greatly contribute to elucidating strategies for controlling arbovirus transmission. This work investigated the infection of guts from Aedes aegypti mosquitoes in an ex vivo platform. We found several MBVs capable of replicating in the gut tissue, including viruses of major health importance, such as dengue, chikungunya, and Zika viruses. In addition, antiviral compounds reduced arbovirus infection in the cultured gut tissue. Overall, the gut model emerges as a useful tool for diverse applications such as studying tissue-specific responses to virus infection and screening potential anti-arboviral molecules.

ABSTRACT Infected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: resistance, used to abrogate virus replication, and disease tolerance, used to avoid tissue damage without controlling viral burden. The principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of bluetongue disease of ruminants, as a model system to investigate the mechanisms of virus-host interactions correlating with disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Using primary cells from multiple donors, we show that BTV consistently reaches higher titers in ovine cells than cells from cattle. The variable replication kinetics of BTV in sheep and cow cells were mostly abolished by abrogating the cell type I interferon (IFN) response. We identified restriction factors blocking BTV replication, but both the sheep and cow orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells than cow cells, which results in an earlier downregulation of antiviral proteins. Moreover, by using RNA sequencing (RNA-seq), we also show a more pronounced expression of interferon-stimulated genes (ISGs) in BTV-infected cow cells than sheep cells. Our data provide a new perspective on how the type I IFN response in reservoir species can have overall positive effects on both virus and host evolution. IMPORTANCE The host immune response usually aims to inhibit virus replication in order to avoid cell damage and disease. In some cases, however, the infected host avoids the deleterious effects of infection despite high levels of viral replication. This strategy is known as disease tolerance, and it is used by animal reservoirs of some zoonotic viruses. Here, using a virus of ruminants (bluetongue virus [BTV]) as an experimental system, we dissected virus-host interactions in cells collected from species that are susceptible (sheep) or tolerant (cow) to disease. We show that (i) virus modulation of the host antiviral type I interferon (IFN) responses, (ii) viral replication kinetics, and (iii) virus-induced cell damage differ in tolerant and susceptible BTV-infected cells. Understanding the complex virus-host interactions in disease tolerance can allow us to disentangle the critical balance between protective and damaging host immune responses.

ABSTRACT Arboviruses are among emerging pathogens of public and veterinary health significance. However, in most of sub-Saharan Africa, their role in the aetiologies of diseases in farm animals is poorly described due to paucity of active surveillance and appropriate diagnosis. Here, we report the discovery of a previously unknown orbivirus in cattle collected in the Kenyan Rift Valley in 2020 and 2021. We isolated the virus in cell culture from the serum of a clinically sick cow aged 2 to 3 years, presenting signs of lethargy. High-throughput sequencing revealed an orbivirus genome architecture with 10 double-stranded RNA segments and a total size of 18,731 bp. The VP1 (Pol) and VP3 (T2) nucleotide sequences of the detected virus, tentatively named Kaptombes virus (KPTV), shared maximum similarities of 77.5% and 80.7% to the mosquito-borne Sathuvachari virus (SVIV) found in some Asian countries, respectively. Screening of 2,039 sera from cattle, goats, and sheep by specific RT-PCR identified KPTV in three additional samples originating from different herds collected in 2020 and 2021. Neutralizing antibodies against KPTV were found in 6% of sera from ruminants (12/200) collected in the region. In vivo experiments with new-born and adult mice induced body tremors, hind limb paralysis, weakness, lethargy, and mortality. Taken together, the data suggest the detection of a potentially disease-causing orbivirus in cattle in Kenya. Its impact on livestock, as well as its potential economic damage, needs to be addressed in future studies using targeted surveillance and diagnostics. IMPORTANCE The genus Orbivirus contains several viruses that cause large outbreaks in wild and domestic animals. However, there is little knowledge on the contribution of orbiviruses to diseases in livestock in Africa. Here, we report the identification of a novel presumably disease-causing orbivirus in cattle, Kenya. The virus, designated Kaptombes virus (KPTV), was initially isolated from a clinically sick cow aged 2 to 3 years, presenting signs of lethargy. The virus was subsequently detected in three additional cows sampled in neighboring locations in the subsequent year. Neutralizing antibodies against KPTV were found in 10% of cattle sera. Infection of new-born and adult mice with KPTV caused severe symptoms and lead to death. Together, these findings indicate the presence of a previously unknown orbivirus in ruminants in Kenya. These data are of relevance as cattle represents an important livestock species in farming industry and often is the main source of livelihoods in rural areas of Africa.

ABSTRACT The primary route of Zika virus (ZIKV) transmission is through the bite of an infected Aedes mosquito, when it probes the skin of a vertebrate host during a blood meal. Viral particles are injected into the bite site together with mosquito saliva and a complex mixture of other components. Some of them are known to play a key role in the augmentation of the arbovirus infection in the host, with increased viremia and/or morbidity. This vector-derived contribution to the infection is not usually considered when vaccine candidates are tested in preclinical animal models. In this study, we performed a preclinical validation of a promising ZIKV vaccine candidate in a mosquito-mouse transmission model using both Asian and African ZIKV lineages. Mice were immunized with engineered ZIKV virus-like particles and subsequently infected through the bite of ZIKV-infected Aedes aegypti mosquitoes. Despite a mild increase in viremia in mosquito-infected mice compared to those infected through traditional needle injection, the vaccine protected the animals from developing the disease and strongly reduced viremia. In addition, during peak viremia, naive mosquitoes were allowed to feed on infected vaccinated and nonvaccinated mice. Our analysis of viral titers in mosquitos showed that the vaccine was able to inhibit virus transmission from the host to the vector. IMPORTANCE Zika is a mosquito-borne viral disease, causing acute debilitating symptoms and complications in infected individuals and irreversible neuronal abnormalities in newborn children. The primary vectors of ZIKV are Aedes aegypti mosquitoes. Despite representing a significant public health burden with a widespread transmission in many regions of the world, Zika remains a neglected disease with no effective antiviral therapies or approved vaccines. It is known that components of the mosquito bite lead to an enhancement of viral infection and spread, but this aspect is often overlooked when vaccine candidates undergo preclinical validation. In this study, we included mosquitoes as viral vectors, demonstrating the ability of a promising vaccine candidate to protect animals against ZIKV infections after the bite of an infected mosquito and to also prevent its further transmission. These findings represent an additional crucial step for the development of an effective prevention tool for clinical use.

ABSTRACT The molecular evolutionary mechanisms underpinning virus-host interactions are increasingly recognized as key drivers of virus emergence, host specificity, and the likelihood that viruses can undergo a host shift that alters epidemiology and transmission biology. Zika virus (ZIKV) is mainly transmitted between humans by Aedes aegypti mosquitoes. However, the 2015 to 2017 outbreak stimulated discussion regarding the role of Culex spp. mosquitoes in transmission. Reports of ZIKV-infected Culex mosquitoes, in nature and under laboratory conditions, resulted in public and scientific confusion. We previously found that Puerto Rican ZIKV does not infect colonized Culex quinquefasciatus, Culex pipiens, or Culex tarsalis, but some studies suggest they may be competent ZIKV vectors. Therefore, we attempted to adapt ZIKV to Cx. tarsalis by serially passaging virus on cocultured Ae. aegypti (Aag2) and Cx. tarsalis (CT) cells to identify viral determinants of species specificity. Increasing fractions of CT cells resulted in decreased overall virus titer and no enhancement of Culex cell or mosquito infection. Next-generation sequencing of cocultured virus passages revealed synonymous and nonsynonymous variants throughout the genome that arose as CT cell fractions increased. We generated nine recombinant ZIKVs containing combinations of the variants of interest. None of these viruses showed increased infection of Culex cells or mosquitoes, demonstrating that variants associated with passaging were not specific to increased Culex infection. These results reveal the challenge of a virus adapting to a new host, even when pushed to adapt artificially. Importantly, they also demonstrate that while ZIKV may occasionally infect Culex mosquitoes, Aedes mosquitoes likely drive transmission and human risk. IMPORTANCE ZIKV is mainly transmitted between humans by Aedes mosquitoes. In nature, ZIKV-infected Culex mosquitoes have been found, and ZIKV infrequently infects Culex mosquitoes under laboratory conditions. Yet, most studies show that Culex mosquitoes are not competent vectors for ZIKV. We attempted to adapt ZIKV to Culex cells to identify viral determinants of species specificity. We sequenced ZIKV after it was passaged on a mixture of Aedes and Culex cells and found that it acquired many variants. We generated recombinant viruses containing combinations of the variants of interest to determine if any of these changes enhance infection in Culex cells or mosquitoes. Recombinant viruses did not show increased infection in Culex cells or mosquitoes, but some variants increased infection in Aedes cells, suggesting adaptation to those cells instead. These results reveal that arbovirus species specificity is complex, and that virus adaptation to a new genus of mosquito vectors likely requires multiple genetic changes.

Oropouche virus (OROV; genus Orthobunyavirus) is the etiological agent of Oropouche fever, a debilitating febrile illness common in South America. We used recombinant expression of the OROV M polyprotein, which encodes the surface glycoproteins Gn and Gc plus the nonstructural protein NSm, to probe the cellular determinants for OROV assembly and budding. Gn and Gc self-assemble and are secreted independently of NSm. Mature OROV Gn has two predicted transmembrane domains that are crucial for glycoprotein translocation to the Golgi complex and glycoprotein secretion, and unlike related orthobunyaviruses, both transmembrane domains are retained during Gn maturation. Disruption of Golgi function using the drugs brefeldin A and monensin inhibits glycoprotein secretion. Infection studies have previously shown that the cellular endosomal sorting complexes required for transport (ESCRT) machinery is recruited to Golgi membranes during OROV assembly and that ESCRT activity is required for virus secretion. A dominant-negative form of the ESCRT-associated ATPase VPS4 significantly reduces recombinant OROV glycoprotein secretion and blocks virus release from infected cells, and VPS4 partly colocalizes with OROV glycoproteins and membranes costained with Golgi markers. Furthermore, immunoprecipitation and fluorescence microscopy experiments demonstrate that OROV glycoproteins interact with the ESCRT-III component CHMP6, with overexpression of a dominant-negative form of CHMP6 significantly reducing OROV glycoprotein secretion. Taken together, our data highlight differences in M polyprotein processing across orthobunyaviruses, indicate that Golgi and ESCRT function are required for glycoprotein secretion, and identify CHMP6 as an ESCRT-III component that interacts with OROV glycoproteins. IMPORTANCE Oropouche virus causes Oropouche fever, a debilitating illness common in South America that is characterized by high fever, headache, myalgia, and vomiting. The tripartite genome of this zoonotic virus is capable of reassortment, and there have been multiple epidemics of Oropouche fever in South America over the last 50 years, making Oropouche virus infection a significant threat to public health. However, the molecular characteristics of this arbovirus are poorly understood. We developed a recombinant protein expression system to investigate the cellular determinants of OROV glycoprotein maturation and secretion. We show that the proteolytic processing of the M polypeptide, which encodes the surface glycoproteins (Gn and Gc) plus a nonstructural protein (NSm), differs between OROV and its close relative Bunyamwera virus. Furthermore, we demonstrate that OROV M glycoprotein secretion requires the cellular endosomal sorting complexes required for transport (ESCRT) membrane-remodeling machinery and identify that the OROV glycoproteins interact with the ESCRT protein CHMP6. [ABSTRACT FROM AUTHOR]

ABSTRACT Usutu virus (USUV, Flaviviridae) is an emerging mosquito-borne virus that has been implicated in neuroinvasive disease in humans and epizootic deaths in wild birds. USUV is maintained in an enzootic cycle between ornithophilic mosquitoes, primarily Culex spp., and wild birds, predominantly passerine species. However, limited experimental data exist on the species competent for USUV transmission. Here, we demonstrate that house sparrows are susceptible to multiple USUV strains. Our study also revealed that Culex quinquefasciatus mosquitoes are susceptible to USUV, with a significantly higher infection rate for the Netherlands 2016 USUV strain compared to the Uganda 2012 USUV strain at 50% and 19%, respectively. To assess transmission between avian host and mosquito vector, we allowed mosquitoes to feed on either juvenile chickens or house sparrows inoculated with USUV. Both bird models transmitted USUV to C. quinquefasciatus mosquitoes. Linear regression analyses indicated that C. quinquefasciatus infection rates were positively correlated with avian viremia levels, with 3 to 4 log10 PFU/mL representing the minimum avian viremia threshold for transmission to mosquitoes. Based on the viremia required for transmission, house sparrows were estimated to more readily transmit the Netherlands 2016 strain compared to the Uganda 2012 strain. These studies provide insights on a competent reservoir host of USUV. IMPORTANCE Usutu virus (USUV) is a zoonotic mosquito-borne virus that can cause neuroinvasive disease, including meningitis and encephalitis, in humans and has resulted in hundreds of thousands of deaths in wild birds. The perpetuation of USUV in nature is dependent on transmission between Culex spp. mosquitoes and various avian species. To date, few experimental data exist for determining which bird species are important for the maintenance of USUV. Our studies showed that house sparrows can transmit infectious Usutu virus, indicating their role as a competent host species. By identifying reservoir species of USUV, we can predict areas of USUV emergence and mitigate its impacts on global human and wildlife health.

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is found in regions where dengue (DENV) and chikungunya (CHIKV) viruses are endemic. Any serological cross-reactivity between DENV, CHIKV, and SARS-CoV-2 is significant as it could lead to misdiagnosis, increased severity, or cross-protection. This study examined the potential cross-reactivity of anti-DENV and CHIKV antibodies with SARS-CoV-2 using acute and convalescent-phase samples collected before the SARS-CoV-2 pandemic. These included healthy, normal human (NHS, n = 6), CHIKV-positive (n = 14 pairs acute and convalescent), primary DENV-positive (n = 20 pairs), secondary DENV-positive (n = 20 pairs), and other febrile illnesses sera (n = 23 pairs). Samples were tested using an in-house SARS-CoV-2 and a EUROIMMUN IgA and IgG ELISAs. All NHS samples were negative, whereas 3.6% CHIKV, 21.7% primary DENV, 15.7% secondary DENV, and 10.8% febrile diseases sera resulted as anti-SARS-CoV-2 antibody positive. The EUROIMMUN ELISA using spike 1 as the antigen detected more positives among the primary DENV infections than the in-house ELISA using spike 1-receptor binding domain (RBD) protein. Among ELISA-positive samples, four had detectable neutralizing antibodies against SARS-CoV-2 reporter virus particles yet none had detectable neutralizing antibodies against the live Wuhan strain of SARS-CoV-2. These data demonstrated the SARS-CoV-2 diagnostic cross-reactivity, but not neutralizing antibody cross-reactivity, among dengue seropositive cases. IMPORTANCE SARS-CoV-2 continues to cause significant morbidity globally, including in areas where DENV and CHIKV are endemic. Reports using rapid diagnostic and ELISAs have demonstrated that serological cross-reactivity between DENV and SARS-CoV-2 can occur. Furthermore, it has been observed that convalescent DENV patients are at a lower risk of developing COVID-19. This phenomenon can interfere with the accuracy of serological testing and clinical management of both DENV and COVID-19 patients. In this study, the cross-reactivity of primary/secondary anti-DENV, CHIKV, and other febrile illness antibodies with SARS-CoV-2 using two ELISAs has been shown. Among ELISA-positive samples, four had detectable levels of neutralizing antibodies against SARS-CoV-2 reporter virus particles. However, none had detectable neutralizing antibodies against the live Wuhan strain of SARS-CoV-2. These data demonstrated SARS-CoV-2 diagnostic cross-reactivity, but not neutralizing antibody cross-reactivity, among dengue seropositive cases. The data discussed here provide information regarding diagnosis and may help guide appropriate public health interventions.

ABSTRACT Mosquitoes are important vectors for many arboviruses. It is becoming increasingly clear that various symbiotic microorganisms (including bacteria and insect-specific viruses; ISVs) in mosquitoes have the potential to modulate the ability of mosquitoes to transmit arboviruses. In this study, we compared the bacteriome and virome (both eukaryotic viruses and bacteriophages) of female adult Aedes aegypti and Culex quinquefasciatus mosquitoes fed with sucrose/water, blood, or blood spiked with Zika virus (ZIKV) or West Nile virus (WNV), respectively. Furthermore, we investigated associations between the microbiota and vector competence. We show that the influence of arboviruses on the mosquito microbiome—and vice versa—is distinct for each combination of arbovirus/mosquito species. The presence of ZIKV resulted in a temporarily increased Aedes ISV diversity. However, this effect was distinct for different ISVs: some ISVs decreased following the blood meal (Aedes aegypti totivirus), whereas other ISVs increased only when the blood contained ZIKV (Guadeloupe mosquito virus). Also, the diversity of the Aedes bacteriome depended on the diet and the presence of ZIKV, with a lower diversity observed for mosquitoes receiving blood without ZIKV. In Cx. quinquefasciatus, some ISVs increased in WNV-infected mosquitoes (Guadeloupe Culex tymo-like virus). Particularly, the presence of Wenzhou sobemo-like virus 3 (WSLV3) was associated with the absence of infectious WNV in mosquito heads, suggesting that WSLV3 might affect vector competence for WNV. Distinct profiles of bacteriophages were identified in Culex mosquitoes depending on diet, despite the lack of clear changes in the bacteriome. Overall, our data demonstrate a complex three-way interaction among arboviruses, resident microbiota, and the host, which is distinct for different arbovirus–mosquito combinations. A better understanding of these interactions may lead to the identification of microbiota able to suppress the ability of arbovirus transmission to humans, and hence improved arbovirus control measures. IMPORTANCE In this study, we first utilized the single mosquito microbiome analysis, demonstrating a complex three-way interaction among arboviruses, resident microbiota, and the host, which is distinct for different arbovirus–mosquito combinations. Some of the previously described “core virus” increased in the mosquitos receiving viral blood meal, like Guadeloupe mosquito virus and Guadeloupe Culex tymo-like virus, suggesting their potential roles in ZIKV and WNV infection. Notably, Wenzhou sobemo-like virus 3 was associated with the absence of infectious WNV in heads of Culex mosquitoes, which might affect vector competence for WNV. A better understanding of these interactions will lead to the identification of microbiota able to suppress the ability of arbovirus transmission to humans, and hence improved arbovirus control measures.

ABSTRACT Zika virus (ZIKV), a mosquito-borne human pathogen, causes dire congenital brain developmental abnormalities in children of infected mothers. The global health crisis precipitated by this virus has led to a concerted effort to develop effective therapies and prophylactic measures although, unfortunately, not very successfully. The error-prone nature of RNA viral genome replication tends to promote evolution of novel viral strains, which could cause epidemics and pandemics. As such, our objective was to develop a safe and effective replication-deficient ZIKV vector-based vaccine candidate. We approached this by generating a ZIKV vector containing only the nonstructural (NS) 5′-untranslated (UTR)-NS-3′ UTR sequences, with the structural proteins capsid (C), precursor membrane (prM), and envelope (E) (CprME) used as a packaging system. We efficiently packaged replication-deficient Zika vaccine particles in human producer cells and verified antigen expression in vitro. In vivo studies showed that, after inoculation in neonatal mice, the Zika vaccine candidate (ZVAX) was safe and did not produce any replication-competent revertant viruses. Immunization of adult, nonpregnant mice showed that ZVAX protected mice from lethal challenge by limiting viral replication. We then evaluated the safety and efficacy of ZVAX in pregnant mice, where it was shown to provide efficient maternal and fetal protection against Zika disease. Mass cytometry analysis showed that vaccinated pregnant animals had high levels of splenic CD8+ T cells and effector memory T cell responses with reduced proinflammatory cell responses, suggesting that endogenous expression of NS proteins by ZVAX induced cellular immunity against ZIKV NS proteins. We also investigated humoral immunity against ZIKV, which is potentially induced by viral proteins present in ZVAX virions. We found no significant difference in neutralizing antibody titer in vaccinated or unvaccinated challenged animals; therefore, it is likely that cellular immunity plays a major role in ZVAX-mediated protection against ZIKV infection. In conclusion, we demonstrated ZVAX as an effective inducer of protective immunity against ZIKV, which can be further evaluated for potential prophylactic application in humans. IMPORTANCE This research is important as it strives to address the critical need for effective prophylactic measures against the outbreak of Zika virus (ZIKV) and outlines an important vaccine technology that could potentially be used to induce immune responses against other pandemic-potential viruses.

ABSTRACT Flaviviruses are important human pathogens worldwide. Diagnostic testing for these viruses is difficult because many of the pathogens require specialized biocontainment. To address this issue, we generated 39 virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells of 13 different flaviviruses, including dengue, yellow fever, Japanese encephalitis, West Nile, St. Louis encephalitis, Zika, Rocio, Ilheus, Usutu, and Powassan viruses. Antigen secretion was stable for at least 10 cell passages, as measured by enzyme-linked immunosorbent assays and immunofluorescence assays. Thirty-five cell lines (90%) had stable antigen expression over 10 passages, with three of these cell lines (7%) increasing in antigen expression and one cell line (3%) decreasing in antigen expression. Antigen secretion in the HEK-293 cell lines was higher than in previously developed COS-1 cell line counterparts. These antigens can replace current antigens derived from live or inactivated virus for safer use in diagnostic testing. IMPORTANCE Serological diagnostic testing for flaviviral infections is hindered by the need for specialized biocontainment for preparation of reagents and assay implementation. The use of previously developed COS-1 cell lines secreting noninfectious recombinant viral antigen is limited due to diminished antigen secretion over time. Here, we describe the generation of 39 flaviviral virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells representing 13 medically important flaviviruses. Antigen production was more stable and statistically higher in these newly developed cell lines than in their COS-1 cell line counterparts. The use of these cell lines for production of flaviviral antigens will expand serological diagnostic testing of flaviviruses worldwide.

Thogotoviruses are tick-borne arboviruses that comprise a unique genus within the Orthomyxoviridae family. Infections with thogotoviruses primarily cause disease in livestock with occasional reports of human infections suggesting a zoonotic potential. In the past, multiple genetically distinct thogotoviruses were isolated mostly from collected ticks. However, many aspects regarding their phylogenetic relationships, morphological characteristics, and virulence in mammals remain unclear. For the present comparative study, we used a collection of 10 different thogotovirus isolates from different geographic areas. Next-generation sequencing and subsequent phylogenetic analyses revealed a distinct separation of these viruses into two major clades, the Thogoto-like and Dhori-like viruses. Electron microscopy demonstrated a heterogeneous morphology with spherical and filamentous particles being present in virus preparations. To study their pathogenicity, we analyzed the viruses in a small animal model system. In intraperitoneally infected C57BL/6 mice, all isolates showed a tropism for liver, lung, and spleen. Importantly, we did not observe horizontal transmission to uninfected, highly susceptible contact mice. The isolates enormously differed in their capacity to induce disease, ranging from subclinical to fatal outcomes. In vivo multistep passaging experiments of two low-pathogenic isolates showed no increased virulence and sequence analyses of the passaged viruses indicated a high stability of the viral genomes after 10 mouse passages. In summary, our analysis demonstrates the broad genetic and phenotypic variability within the thogotovirus genus. Moreover, thogotoviruses are well adapted to mammals but their horizontal transmission seems to depend on ticks as their vectors. IMPORTANCE Since their discovery over 60 years ago, 15 genetically distinct members of the thogotovirus genus have been isolated. These arboviruses belong to the Orthomyxovirus family and share many features with influenza viruses. However, numerous of these isolates have not been characterized in depth. In the present study, we comparatively analyzed a collection of 10 different thogotovirus isolates to answer basic questions about their phylogenetic relationships, morphology, and pathogenicity in mice. Our results highlight shared and unique characteristics of this diverse genus. Taken together, these observations provide a framework for the phylogenic classification and phenotypic characterization of newly identified thogotovirus isolates that could potentially cause severe human infections as exemplified by the recently reported, fatal Bourbon virus cases in the United States. [ABSTRACT FROM AUTHOR]

ABSTRACT Arboviruses transmitted by mosquitoes are responsible for the death of millions of people each year. In addition to arboviruses, many insect-specific viruses (ISVs) have been discovered in mosquitoes in the last decade. ISVs, in contrast to arboviruses transmitted by mosquitoes to vertebrates, cannot replicate in vertebrate cells even when they are evolutionarily closely related to arboviruses. The alphavirus genus includes many arboviruses, although only a few ISVs have been discovered from this genus so far. Here, we investigate the interactions of a recently isolated insect-specific alphavirus, Agua Salud alphavirus (ASALV), with its mosquito host. RNA interference (RNAi) is one of the essential antiviral responses against arboviruses, although there is little knowledge on the interactions of RNAi with ISVs. Through the knockdown of transcripts of the different key RNAi pathway (small interfering RNA [siRNA], microRNA [miRNA], and P-element-induced wimpy testis [PIWI]-interacting RNA [piRNA]) proteins, we show the antiviral role of Ago2 (siRNA), Ago1 (miRNA), and Piwi4 proteins against ASALV in Aedes aegypti-derived cells. ASALV replication was increased in Dicer2 and Ago2 knockout cells, confirming the antiviral role of the siRNA pathway. In infected cells, mainly ASALV-specific siRNAs are produced, while piRNA-like small RNAs, with the characteristic nucleotide bias resulting from ping-pong amplification, are produced only in Dicer2 knockout cells. Taken together, ASALV interactions with the mosquito RNAi response differ from those of arthropod-borne alphaviruses in some aspects, although they also share some commonalities. Further research is needed to understand whether the identified differences can be generalized to other insect-specific alphaviruses. IMPORTANCE Mosquitoes are efficient vectors for many arboviruses that cause emergent infectious diseases in humans. Many insect-specific viruses (ISVs) that can infect mosquitoes but cannot infect vertebrates have been discovered in the last decade. ISVs have attracted great attention due to their potential use in mosquito or arbovirus control, by either decreasing mosquito fitness or restricting arbovirus replication and transmission to humans. However, ISV-mosquito interactions are not well understood. RNA interference (RNAi) is the most important innate immune response against many arboviruses, while it is unknown if it is antiviral against ISVs. Here, we investigate in detail the antiviral effect of the RNAi response in mosquitoes against an ISV for the first time. Using a recently isolated insect-specific alphavirus, we show that the regulation of virus replication was different from that for arthropod-borne alphaviruses despite some similarities. The differences in mosquito-virus interactions could drive the different transmission modes, which could eventually drive the evolution of arboviruses. Hence, an understanding of mosquito-ISV interactions can shed light on the ecology and evolution of both ISVs and the medically important arboviruses.

ABSTRACT Arthropod-borne viruses comprise a significant global disease burden. Surveillance and mitigation of arboviruses like Zika virus (ZIKV) require accurate estimates of transmissibility by vector mosquitoes. Although Aedes species mosquitoes are established as competent ZIKV vectors, differences in experimental protocols across studies prevent direct comparisons of relative transmissibility. An understudied factor complicating these comparisons is differential environmental microbiota exposures, where most vector competence studies use mosquitoes reared in laboratory tap water, which does not represent the microbial complexity of environmental water where wild larvae develop. We simulated natural larval development by rearing Californian Aedes aegypti larvae with microbes obtained from cemetery headstone water compared to conventional tap water. A. aegypti larvae reared in environmental cemetery water pupated 3 days faster and at higher rates. Mosquitoes reared in environmental water were less competent vectors of ZIKV than laboratory water-reared A. aegypti, as evidenced by significantly reduced infection and transmission rates. Microbiome comparisons of laboratory water- and environment water-reared mosquitoes and their rearing water showed significantly higher bacterial diversity in environment water. Despite this pattern, corresponding differences in bacterial diversity were not consistently observed between the respective adult mosquitoes. We also observed that the microbial compositions of adult mosquitoes differed more by whether they ingested a bloodmeal than by larval water type. Together, these results highlight the role of transient microbes in the larval environment in modulating A. aegypti vector competence for ZIKV. Laboratory vector competence likely overestimates the true transmissibility of arboviruses like ZIKV when conventional laboratory water is used for rearing. IMPORTANCE We observed that A. aegypti mosquitoes reared in water from cemetery headstones instead of the laboratory tap exhibited a reduced capacity to become infected with and transmit Zika virus. Water from the environment contained more bacterial species than tap water, but these bacteria were not consistently detected in adult mosquitoes. Our results suggest that rearing mosquito larvae in water collected from local environments as opposed to laboratory tap water, as is conventional, could provide a more realistic assessment of ZIKV vector competence since it better recapitulates the natural environment in which larvae develop. Given that laboratory vector competence is used to define the species to target for control, the use of environmental water to rear larvae could better approximate the microbial exposures of wild mosquitoes, lessening the potential for overestimating ZIKV transmission risk. These studies raise the question of whether rearing larvae in natural water sources also reduces vector competence for other mosquito-borne viruses.

ABSTRACT Previous studies have shown that the adaptation of Indian Ocean lineage (IOL) chikungunya virus (CHIKV) strains for Aedes albopictus transmission was mediated by an E1-A226V substitution, followed by either a single substitution in E2 or synergistic substitutions in the E2 and E3 envelope glycoproteins. Here, we examined whether Asian lineage strains, including those that descended from the 2014 Caribbean introduction, are likely to acquire these A. albopictus-adaptive E2 substitutions. Because Asian lineage strains cannot adapt through the E1-A226V substitution due to an epistatic constraint, we first determined that the beneficial effect of these E2 mutations in IOL strains is independent of E1-A226V. We then introduced each of these E2 adaptive mutations into the Asian lineage backbone to determine if they improve infectivity for A. albopictus. Surprisingly, our results indicated that in the Asian lineage backbone, these E2 mutations significantly decreased CHIKV fitness in A. albopictus. Furthermore, we tested the effects of these mutations in Aedes aegypti and observed different results from those in A. albopictus, suggesting that mosquito species-specific factors that interact with the envelope proteins are involved in vector infection efficiency. Overall, our results indicate that the divergence between Asian lineage and IOL CHIKVs has led them onto different adaptive landscapes with differing potentials to expand their vector host range. IMPORTANCE Since its introduction into the Caribbean in October 2013, CHIKV has rapidly spread to almost the entire neotropical region. However, its potential to further spread globally, including into more temperate climates, depends in part on its ability to be transmitted efficiently by Aedes albopictus, which can survive colder winters than A. aegypti. We examined in an Asian lineage backbone A. albopictus-adaptive mutations that arose from 2005 to 2009 in Indian Ocean lineage (IOL) strains. Our results predict that the Asian CHIKV lineage now in the Americas will not readily adapt for enhanced A. albopictus transmission via the same mechanisms or adaptive mutations used previously by IOL strains. The vector species- and CHIKV lineage-specific effects caused by adaptive CHIKV envelope glycoprotein substitutions may elucidate our understanding of the mechanisms of mosquito infection and spread.

As climate change alters Earth's biomes, it is expected the transmission dynamics of mosquito-borne viruses will change. While the effects of temperature changes on mosquito-virus interactions and the spread of the pathogens have been elucidated over the last decade, the impact of relative humidity changes is still relatively unknown. To overcome this knowledge gap, we exposed Aedes aegypti females to various humidity conditions. We measured different components of vectorial capacity such as survival, blood-feeding rates, and changes in infection and dissemination of Zika virus. Survival decreased as the humidity level decreased, while infection rates increased as the humidity level decreased. Alternatively, blood feeding rates and disseminated infection rates peaked at the intermediate 50% relative humidity treatment but were the same in the 30% and 80% relative humidity treatments. These results provide empirical evidence that Ae. aegypti exposure to low humidity can enhance Zika virus infection in the mosquito, which has important implications in predicting how climate change will impact mosquito-borne viruses.IMPORTANCEViruses transmitted by mosquitoes to humans are a major public health burden and are expected to increase under climate change. While we know that temperature is an important driver of variation in arbovirus replication in the mosquito, very little is known about how other relevant climate variables such as humidity will influence the interaction between mosquitoes and the viruses they transmit. Given the variability in humidity across environments, and the predicted changes in humidity under climate change, it is imperative that we also study the impact that it has on mosquito infection and transmission of arboviruses., Competing Interests: The authors declare no conflict of interest.

ABSTRACT Oropouche virus (OROV) infection of humans is associated with a debilitating febrile illness that can progress to meningitis or encephalitis. First isolated from a forest worker in Trinidad and Tobago in 1955, the arbovirus OROV has since been detected throughout the Amazon basin with an estimated 500,000 human infections over 60 years. Like other members of the family Peribunyaviridae, the viral genome exists as 3 single-stranded negative-sense RNA segments. The medium-sized segment encodes a viral glycoprotein complex (GPC) that is proteolytically processed into two viral envelope proteins, Gn and Gc, responsible for attachment and membrane fusion. There are no therapeutics or vaccines to combat OROV infection, and we have little understanding of protective immunity to infection. Here, we generated a replication competent chimeric vesicular stomatitis virus (VSV), in which the endogenous glycoprotein was replaced by the GPC of OROV. Serum from mice immunized by intramuscular injection with VSV-OROV specifically neutralized wild-type OROV, and using peptide arrays we mapped multiple epitopes within an N-terminal variable region of Gc recognized by the immune sera. VSV-OROV lacking this variable region of Gc was also immunogenic in mice producing neutralizing sera that recognize additional regions of Gc. Challenge of both sets of immunized mice with wild-type OROV shows that the VSV-OROV chimeras reduce wild-type viral infection and suggest that antibodies that recognize the variable N terminus of Gc afford less protection than those that target more conserved regions of Gc. IMPORTANCE Oropouche virus (OROV), an orthobunyavirus found in Central and South America, is an emerging public health challenge that causes debilitating febrile illness. OROV is transmitted by arthropods, and increasing mobilization has the potential to significantly increase the spread of OROV globally. Despite this, no therapeutics or vaccines have been developed to combat infection. Using vesicular stomatitis (VSV) as a backbone, we developed a chimeric virus bearing the OROV glycoproteins (VSV-OROV) and tested its ability to elicit a neutralizing antibody response. Our results demonstrate that VSV-OROV produces a strong neutralizing antibody response that is at least partially targeted to the N-terminal region of Gc. Importantly, vaccination with VSV-OROV reduces viral loads in mice challenged with wild-type virus. These data provide novel evidence that targeting the OROV glycoproteins may be an effective vaccination strategy to combat OROV infection.

Recent field trials have demonstrated that dengue incidence can be substantially reduced by introgressing strains of the endosymbiotic bacterium Wolbachia into Aedes aegypti mosquito populations. This strategy relies on Wolbachia reducing the susceptibility of Ae. aegypti to disseminated infection by positive-sense RNA viruses like dengue. However, RNA viruses are well known to adapt to antiviral pressures. Here, we review the viral infection stages where selection for Wolbachia-resistant virus variants could occur. We also consider the genetic constraints imposed on viruses that alternate between vertebrate and invertebrate hosts, and the likely selection pressures to which dengue virus might adapt in order to be effectively transmitted by Ae. aegypti that carry Wolbachia. While there are hurdles to dengue viruses developing resistance to Wolbachia, we suggest that long-term surveillance for resistant viruses should be an integral component of Wolbachia-introgression biocontrol programs. [ABSTRACT FROM AUTHOR]

Arboviruses are transmitted by specific vectors, and the reasons for this specificity are not fully understood. One contributing factor is the existence of tissue barriers within the vector such as the midgut escape barrier. We used microRNA (miRNA) targeting of Sindbis virus (SINV) to study how replication in midgut cells contributes to overcoming this barrier in the mosquito Aedes aegypti. SINV constructs were designed to be attenuated specifically in midgut cells by inserting binding sites for midgut-specific miRNAs into either the 39 untranslated region (MRE39miRT) or the structural open reading frame (MRE-ORFmiRT) of the SINV genome. Both miRNA-targeted viruses replicated less efficiently than control viruses in the presence of these miRNAs. When mosquitoes were given infectious blood meals containing miRNA-targeted viruses, only around 20% (MRE39miRT) or 40% (MREORFmiRT) of mosquitoes developed disseminated infection. In contrast, dissemination occurred in almost all mosquitoes fed control viruses. Deep sequencing of virus populations from individual mosquitoes ruled out selection for mutations in the inserted target sequences as the cause for dissemination in these mosquitoes. In mosquitoes that became infected with miRNA-targeted viruses, titers were equivalent to those of mosquitoes infected with control virus in both the midgut and the carcass, and there was no evidence of a threshold titer necessary for dissemination. Instead, it appeared that if infection was successfully established in the midgut, replication and dissemination were largely normal. Our results support the hypothesis that replication is an important factor in allowing SINV to overcome the midgut escape barrier but hint that other factors are also likely involved. Arboviruses are transmitted by specific vectors, and the reasons for this specificity are not fully understood. One contributing factor is the existence of tissue barriers within the vector such as the midgut escape barrier. We used microRNA (miRNA) targeting of Sindbis virus (SINV) to study how replication in midgut cells contributes to overcoming this barrier in the mosquito Aedes aegypti. SINV constructs were designed to be attenuated specifically in midgut cells by inserting binding sites for midgut-specific miRNAs into either the 39 untranslated region (MRE39miRT) or the structural open reading frame (MRE-ORFmiRT) of the SINV genome. Both miRNA-targeted viruses replicated less efficiently than control viruses in the presence of these miRNAs. When mosquitoes were given infectious blood meals containing miRNA-targeted viruses, only around 20% (MRE39miRT) or 40% (MREORFmiRT) of mosquitoes developed disseminated infection. In contrast, dissemination occurred in almost all mosquitoes fed control viruses. Deep sequencing of virus populations from individual mosquitoes ruled out selection for mutations in the inserted target sequences as the cause for dissemination in these mosquitoes. In mosquitoes that became infected with miRNA-targeted viruses, titers were equivalent to those of mosquitoes infected with control virus in both the midgut and the carcass, and there was no evidence of a threshold titer necessary for dissemination. Instead, it appeared that if infection was successfully established in the midgut, replication and dissemination were largely normal. Our results support the hypothesis that replication is an important factor in allowing SINV to overcome the midgut escape barrier but hint that other factors are also likely involved. [ABSTRACT FROM AUTHOR]

Although fetal death is now understood to be a severe outcome of congenital Zika syndrome, the role of viral genetics is still unclear. We sequenced Zika virus (ZIKV) from a rhesus macaque fetus that died after inoculation and identified a single intrahost substitution, M1404I, in the ZIKV polyprotein, located in nonstructural protein 2B (NS2B). Targeted sequencing flanking position 1404 in 9 additional macaque mothers and their fetuses identified M1404I at a subconsensus frequency in the majority (5 of 9, 56%) of animals and some of their fetuses. Despite its repeated presence in pregnant macaques, M1404I has occurred rarely in humans since 2015. Since the primary ZIKV transmission cycle is human-mosquito-human, mutations in one host must be retained in the alternate host to be perpetuated. We hypothesized that ZIKV I1404 increases viral fitness in nonpregnant macaques and pregnant mice but is less efficiently transmitted by vectors, explaining its low frequency in humans during outbreaks. By examining competitive fitness relative to that of ZIKV M1404, we observed that ZIKV I1404 produced lower viremias in nonpregnant macaques and was a weaker competitor in tissues. In pregnant wild-type mice, ZIKV I1404 increased the magnitude and rate of placental infection and conferred fetal infection, in contrast to ZIKV M1404, which was not detected in fetuses. Although infection and dissemination rates were not different, Aedes aegypti mosquitoes transmitted ZIKV I1404 more poorly than ZIKV M1404. Our data highlight the complexity of arbovirus mutation-fitness dynamics and suggest that intrahost ZIKV mutations capable of augmenting fitness in pregnant vertebrates may not necessarily spread efficiently via mosquitoes during epidemics. [ABSTRACT FROM AUTHOR]

ABSTRACT Sandfly-transmitted phleboviruses (family Phenuiviridae, order Bunyavirales) are associated with febrile illness and infections of the nervous system in humans. These viruses are almost exclusively found in tropical areas of the New World and restricted to semiarid and temperate zones in the Old World. Here, we discovered seven strains of four previously unknown phleboviruses, named Bogoria virus (BOGV), Embossos virus (EMRV), Kiborgoch virus (KBGV), and Perkerra virus (PERV), as well as the recently discovered Ntepes virus, in sandflies collected in the Kenyan Rift Valley. The genomes have a tripartite organization with conserved termini typical of phleboviruses. LOBV, PERV, and EMBV showed low similarity to known phleboviruses, with less than 55% pairwise amino acid identities in the RNA-directed RNA polymerase (RdRp) proteins, and defined a highly diversified monophyletic clade in sister relationship to the sandfly fever Sicilian serocomplex. All three viruses failed to react with sandfly fever Sicilian virus antisera in recombinant immunofluorescence assays (rIFA), suggesting that the viruses belong to a yet-unknown serogroup. In contrast, KBGV was closely related to Toscana virus (84% identity of RdRp proteins) and shared a most recent common ancestor with the clade comprising sandfly fever Naples and Toscana viruses. KBGV reacted with sandfly fever Naples and Toscana virus antisera in rIFA. The genetic diversity of the detected viruses and their phylogenetic positions implies that the Old World sandfly-borne phleboviruses originated from sub-Saharan Africa. Importantly, our findings suggest that diseases associated with sandfly-borne phlebovirus infections may also affect the Kenyan population. IMPORTANCE Studies on the genetic diversity of arthropod-borne viruses circulating in rural regions can provide critical early indications on new emerging viruses essential for global epidemic preparedness. In this study, we describe the discovery of four phleboviruses in sandflies from the Kenyan Rift Valley. The novel viruses are related to the two medically important serocomplexes, sandfly fever Naples and sandfly fever Sicilian, that are associated with febrile illness and neuroinvasive infections and which were previously not known to occur in sub-Saharan Africa. Knowledge on the occurrence of sandfly-borne phleboviruses in Kenya and elsewhere in Africa can help to decipher their contributions in the etiologies of fevers of unknown origin in patients. Our findings on five genetically diverse phleboviruses detected in Kenya suggest that the common ancestor of Old World phleboviruses existed in sub-Saharan Africa, a hot spot for emerging arboviruses.

ABSTRACT Arboviruses are pathogens of humans and animals. A better understanding of the interactions between these pathogens and the arthropod vectors, such as mosquitoes, that transmit them is necessary to develop novel control measures. A major antiviral pathway in the mosquito vector is the exogenous small interfering RNA (exo-siRNA) pathway, which is induced by arbovirus-derived double-stranded RNA in infected cells. Although recent work has shown the key role played by Argonaute-2 (Ago-2) and Dicer-2 (Dcr-2) in this pathway, the regulatory mechanisms that govern these pathways have not been studied in mosquitoes. Here, we show that the Domino ortholog p400 has antiviral activity against the alphavirus Semliki Forest virus (Togaviridae) both in Aedes aegypti-derived cells and in vivo. Antiviral activity of p400 was also demonstrated against chikungunya virus (Togaviridae) and Bunyamwera virus (Peribunyaviridae) but not Zika virus (Flaviviridae). p400 was found to be expressed across mosquito tissues and regulated ago-2 but not dcr-2 transcript levels in A. aegypti mosquitoes. These findings provide novel insights into the regulation of an important aedine exo-siRNA pathway effector protein, Ago-2, by the Domino ortholog p400. They add functional insights to previous observations of this protein’s antiviral and RNA interference regulatory activities in Drosophila melanogaster. IMPORTANCE Female Aedes aegypti mosquitoes are vectors of human-infecting arthropod-borne viruses (arboviruses). In recent decades, the incidence of arthropod-borne viral infections has grown dramatically. Vector competence is influenced by many factors, including the mosquito’s antiviral defenses. The exogenous small interfering RNA (siRNA) pathway is a major antiviral response restricting arboviruses in mosquitoes. While the roles of the effectors of this pathway, Argonaute-2 and Dicer-2 are well characterized, nothing is known about its regulation in mosquitoes. In this study, we demonstrate that A. aegypti p400, whose ortholog Domino in Drosophila melanogaster is a chromatin-remodeling ATPase member of the Tip60 complex, regulates siRNA pathway activity and controls ago-2 expression levels. In addition, we found p400 to have antiviral activity against different arboviruses. Therefore, our study provides new insights into the regulation of the antiviral response in A. aegypti mosquitoes.

Alphaviruses are insect-borne viruses that alternate between replication in mosquitoes and vertebrate species. Adaptation of some alphaviruses to vertebrate hosts has involved the acquisition of an RNA structure (downstream loop [DLP]) in viral subgenomic mRNAs that confers translational resistance to protein kinase (PKR)- mediated eIF2α phosphorylation. Here, we found that, in addition to promoting eIF2-independent translation of viral subgenomic mRNAs, presence of the DLP structure also increased the resistance of alphavirus to type I interferon (IFN). Aura virus (AURAV), an ecologically isolated relative of Sindbis virus (SV) that is poorly adapted to replication in vertebrate cells, displayed a nonfunctional DLP structure and dramatic sensitivity to type I IFN. Our data suggest that an increased resistance to IFN emerged during translational adaptation of alphavirus mRNA to vertebrate hosts, reinforcing the role that double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays as both a constitutive and IFN-induced antiviral effector. Interestingly, a mutant SV lacking the DLP structure (SV-ΔDLP) and AURAV both showed a marked oncotropism for certain tumor cell lines that have defects in PKR expression and/or activation. AURAV selectively replicated in and killed some cell lines derived from human hepatocarcinoma (HCC) that lacked PKR response to infection or poly(I・C) transfection. The oncolytic activities of SV-ΔDLP and AURAV were also confirmed using tumor xenografts in mice, showing tumor regression activities comparable to wild-type SV. Our data show that translation of alphavirus subgenomic mRNAs plays a central role in IFN susceptibility and cell tropism, suggesting an unanticipated oncolytic potential that some naive arboviruses may have in virotherapy. [ABSTRACT FROM AUTHOR]

Ross River virus (RRV), an alphavirus of the Togaviridae family, is the most medically significant mosquito-borne virus of Australia. Past RRV phylogenetic and evolutionary analyses have been based on partial genome analyses only. Three geographically distinct RRV lineages, the Eastern, the Western, and the supposedly extinct North-Eastern lineage, were classified previously. We sought to expand on past phylogenies through robust genome-scale phylogeny to better understand RRV genetic diversity and evolutionary dynamics. We analyzed 106 RRV complete coding sequences, which included 13 genomes available on NCBI and 94 novel sequences derived for this study, sampled throughout Western Australia (1977-2014) and during the substantial Pacific Islands RRV epidemic (1979 -1980). Our final data set comprised isolates sampled over 59 years (1959 -2018) from a range of locations. Four distinct genotypes were defined, with the newly described genotype 4 (G4) found to be the contemporary lineage circulating in Western Australia. The prior geographical classification of RRV lineages was not supported by our findings, with evidence of geographical and temporal cocirculation of distinct genetic groups. Bayesian Markov chain Monte Carlo (MCMC) analysis revealed that RRV lineages diverged from a common ancestor approximately 94 years ago, with distinct lineages emerging roughly every 10 years over the past 50 years in periodic bursts of genetic diversity. Our study has enabled a more robust analysis of RRV evolutionary history and resolved greater genetic diversity that had been previously defined by partial E2 gene analysis. [ABSTRACT FROM AUTHOR]

ABSTRACT Arthropod-borne viruses are diverse pathogens and are often associated with human disease. These viruses span multiple genera, including flaviviruses, alphaviruses, and bunyaviruses. In a high-throughput drug screen, we found that tenovin-1 was antiviral against the flaviviruses Zika virus and dengue virus. Tenovin-1 is a sirtuin inhibitor, and here we found that inhibition of sirtuins, but not inhibition of the related histone deacetylases, is potently antiviral against diverse arboviruses. Sirtuin inhibitors block infection of arboviruses in multiple human cell types. We found that sirtuin inhibitors arrest infection downstream of entry but that they do so at an early step, preventing the accumulation of viral RNA and protein. However, sirtuin inhibitors had no impact on the replication of flaviviral replicons, suggesting a defect in the establishment of replication. Consistent with this, we found that sirtuin inhibitors impacted double-stranded RNA (dsRNA) accumulation during flaviviral infection. Since these viruses infect vector insects, we also tested whether sirtuin inhibitors impacted infection of adult flies and found that these inhibitors blocked infection; therefore, they target highly conserved facets of replication. Taken together, these results suggest that sirtuin inhibitors represent a new class of potent host-targeting antivirals. IMPORTANCE Arthropod-borne viruses are diverse pathogens and are associated with human disease. Through high-throughput drug screening, we found that sirtuin inhibitors are potently antiviral against diverse arboviruses, including flaviviruses such as West Nile virus, bunyaviruses such as Rift Valley fever virus, and alphaviruses such as chikungunya virus. Sirtuin inhibitors block infection of these viruses in multiple human cell types. Moreover, we found that sirtuin inhibitors arrest infection downstream of entry but that they do so at an early step, preventing the accumulation of viral RNA and protein. Since these viruses infect vector insects, we also tested whether sirtuin inhibitors impacted infection of adult flies and found that these inhibitors blocked infection; therefore, they target highly conserved facets of replication. Taken together, these results suggest that sirtuin inhibitors represent a new class of potent host-targeting antivirals.

Wolbachia are a genus of insect endosymbiotic bacteria which includes strains w Mel and w AlbB that are being utilized as a biocontrol tool to reduce the incidence of Aedes aegypti -transmitted viral diseases like dengue. However, the precise mechanisms underpinning the antiviral activity of these Wolbachia strains are not well defined. Here, we generated a panel of Ae. aegypti -derived cell lines infected with antiviral strains w Mel and w AlbB or the non-antiviral Wolbachia strain w Pip to understand host cell morphological changes specifically induced by antiviral strains. Antiviral strains were frequently found to be entirely wrapped by the host endoplasmic reticulum (ER) membrane, while w Pip bacteria clustered separately in the host cell cytoplasm. ER-derived lipid droplets (LDs) increased in volume in w Mel- and w AlbB-infected cell lines and mosquito tissues compared to cells infected with w Pip or Wolbachia -free controls. Inhibition of fatty acid synthase (required for triacylglycerol biosynthesis) reduced LD formation and significantly restored ER-associated dengue virus replication in cells occupied by w Mel. Together, this suggests that antiviral Wolbachia strains may specifically alter the lipid composition of the ER to preclude the establishment of dengue virus (DENV) replication complexes. Defining Wolbachia 's antiviral mechanisms will support the application and longevity of this effective biocontrol tool that is already being used at scale.IMPORTANCE Aedes aegypti transmits a range of important human pathogenic viruses like dengue. However, infection of Ae. aegypti with the insect endosymbiotic bacterium, Wolbachia , reduces the risk of mosquito to human viral transmission. Wolbachia is being utilized at field sites across more than 13 countries to reduce the incidence of viruses like dengue, but it is not well understood how Wolbachia induces its antiviral effects. To examine this at the subcellular level, we compared how different strains of Wolbachia with varying antiviral strengths associate with and modify host cell structures. Strongly antiviral strains were found to specifically associate with the host endoplasmic reticulum and induce striking impacts on host cell lipid droplets. Inhibiting Wolbachia -induced lipid redistribution partially restored dengue virus replication demonstrating this is a contributing role for Wolbachia's antiviral activity. These findings provide new insights into how antiviral Wolbachia strains associate with and modify Ae. aegypti host cells., Competing Interests: The authors declare no conflict of interest.

Importance: The recent Zika virus (ZIKV) epidemic in the Americas highlights its potential public health threat. While the Asian ZIKV lineage has been identified as the main cause of the epidemic, the African lineage, which has been primarily confined to Africa, has shown evidence of higher transmissibility in Aedes mosquitoes. To gain a deeper understanding of this differential transmissibility, our study employed a combination of tissue-level infection kinetics and single-cell-level infection kinetics using in situ immunofluorescent staining. We discovered that the African ZIKV lineage propagates more rapidly and spreads more efficiently within mosquito cells and tissues than its Asian counterpart. This information lays the groundwork for future exploration of the viral and host determinants driving these variations in propagation efficiency., Competing Interests: The authors declare no conflict of interest.

Aedes aegypti mosquitoes are the main vectors of arthropod-borne viruses (arboviruses) of public health significance, such as the flaviviruses dengue virus (DENV) and Zika virus (ZIKV). Mosquitoes are also the natural hosts of a wide range of viruses that are insect specific, raising the question of their influence on arbovirus transmission in nature. Cell-fusing agent virus (CFAV) was the first described insectspecific flavivirus, initially discovered in an A. aegypti cell line and subsequently detected in natural A. aegypti populations. It was recently shown that DENV and the CFAV strain isolated from the A. aegypti cell line have mutually beneficial interactions in mosquito cells in culture. However, whether natural strains of CFAV and DENV interact in live mosquitoes is unknown. Using a wild-type CFAV isolate recently derived from Thai A. aegypti mosquitoes, we found that CFAV negatively interferes with both DENV type 1 and ZIKV in vitro and in vivo. For both arboviruses, prior infection by CFAV reduced the dissemination titer in mosquito head tissues. Our results indicate that the interactions observed between arboviruses and the CFAV strain derived from the cell line might not be a relevant model of the viral interference that we observed in vivo. Overall, our study supports the hypothesis that insect-specific flaviviruses may contribute to reduce the transmission of humanpathogenic flaviviruses. [ABSTRACT FROM AUTHOR]

Arthropod genomes contain sequences derived from integrations of DNA and nonretroviral RNA viruses. These sequences, known as endogenous viral elements (EVEs), have been acquired over the course of evolution and have been proposed to serve as a record of past viral infections. Recent evidence indicates that EVEs can function as templates for the biogenesis of PIWI-interacting RNAs (piRNAs) in some mosquito species and cell lines, raising the possibility that EVEs may serve as a source of immunological memory in these organisms. However, whether piRNAs are derived from EVEs or serve an antiviral function in other arthropod species is unknown. Here, we used publicly available genome assemblies and small RNA sequencing data sets to characterize the repertoire and function of EVEs across 48 arthropod genomes. We found that EVEs are widespread in arthropod genomes and primarily correspond to unclassified single-stranded RNA (ssRNA) viruses and viruses belonging to the Rhabdoviridae and Parvoviridae families. Additionally, EVEs were enriched in piRNA clusters in a majority of species, and we found that production of primary piRNAs from EVEs is common, particularly for EVEs located within piRNA clusters. While the abundance of EVEs within arthropod genomes and the frequency with which EVEs give rise to primary piRNAs generally support the hypothesis that EVEs contribute to an antiviral response via the piRNA pathway, limited nucleotide identity between currently described viruses and EVEs identified here likely limits the extent to which this process plays a role during infection with known viruses in the arthropod species analyzed. [ABSTRACT FROM AUTHOR]

ABSTRACT Mosquito-transmitted viruses are spread globally and present a great risk to human health. Among the many approaches investigated to limit the diseases caused by these viruses are attempts to make mosquitos resistant to virus infection. Coinfection of mosquitos with the bacterium Wolbachia pipientis from supergroup A is a recent strategy employed to reduce the capacity for major vectors in the Aedes mosquito genus to transmit viruses, including dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV). Recently, a supergroup B Wolbachia wStri, isolated from Laodelphax striatellus, was shown to inhibit multiple lineages of ZIKV in Aedes albopictus cells. Here, we show that wStri blocks the growth of positive-sense RNA viruses DENV, CHIKV, ZIKV, and yellow fever virus by greater than 99.9%. wStri presence did not affect the growth of the negative-sense RNA viruses LaCrosse virus or vesicular stomatitis virus. Investigation of the stages of the ZIKV life cycle inhibited by wStri identified two distinct blocks in viral replication. We found a reduction of ZIKV entry into wStri-infected cells. This was partially rescued by the addition of a cholesterol-lipid supplement. Independent of entry, transfected viral genome was unable to replicate in Wolbachia-infected cells. RNA transfection and metabolic labeling studies suggested that this replication defect is at the level of RNA translation, where we saw a 66% reduction in mosquito protein synthesis in wStri-infected cells. This study’s findings increase the potential for application of wStri to block additional arboviruses and also identify specific blocks in viral infection caused by Wolbachia coinfection. IMPORTANCE Dengue, Zika, and yellow fever viruses are mosquito-transmitted diseases that have spread throughout the world, causing millions of infections and thousands of deaths each year. Existing programs that seek to contain these diseases through elimination of the mosquito population have so far failed, making it crucial to explore new ways of limiting the spread of these viruses. Here, we show that introduction of an insect symbiont, Wolbachia wStri, into mosquito cells is highly effective at reducing yellow fever virus, dengue virus, Zika virus, and Chikungunya virus production. Reduction of virus replication was attributable to decreases in entry and a strong block of virus gene expression at the translational level. These findings expand the potential use of Wolbachia wStri to block viruses and identify two separate steps for limiting virus replication in mosquitos that could be targeted via microbes or other means as an antiviral strategy.

ABSTRACT Interferon alpha/beta (IFN-α/β) is a critical mediator of protection against most viruses, with host survival frequently impossible in its absence. Many studies have investigated the pathways involved in the induction of IFN-α/β after virus infection and the resultant upregulation of antiviral IFN-stimulated genes (ISGs) through IFN-α/β receptor complex signaling. However, other than examining the effects of genetic deletion of induction or effector pathway components, little is known regarding the functionality of these responses in intact hosts and whether host genetic or environmental factors might influence their potency. Here, we demonstrate that the IFN-α/β response against multiple arthropod-vectored viruses, which replicate over a wide temperature range, is extremely sensitive to fluctuations in temperature, exhibiting reduced antiviral efficacy at subnormal cellular temperatures and increased efficacy at supranormal temperatures. The effect involves both IFN-α/β and ISG upregulation pathways with a major aspect of altered potency reflecting highly temperature-dependent transcription of IFN response genes that leads to altered IFN-α/β and ISG protein levels. Discordantly, signaling steps prior to transcription that were examined showed the opposite effect from gene transcription, with potentiation at low temperature and inhibition at high temperature. Finally, we demonstrate that by lowering the temperature of mice, chikungunya arbovirus replication and disease are exacerbated in an IFN-α/β-dependent manner. This finding raises the potential for use of hyperthermia as a therapeutic modality for viral infections and in other contexts such as antitumor therapy. The increased IFN-α/β efficacy at high temperatures may also reflect an innate immune-relevant aspect of the febrile response. IMPORTANCE The interferon alpha/beta (IFN-α/β) response is a first-line innate defense against arthropod-borne viruses (arboviruses). Arboviruses, such as chikungunya virus (CHIKV), can infect cells and replicate across a wide temperature range due to their replication in both mammalian/avian and arthropod hosts. Accordingly, these viruses can cause human disease in tissues regularly exposed to temperatures below the normal mammalian core temperature, 37°C. We questioned whether temperature variation could affect the efficacy of IFN-α/β responses against these viruses and help to explain some aspects of human disease manifestations. We observed that IFN-α/β efficacy was dramatically lower at subnormal temperatures and modestly enhanced at febrile temperatures, with the effects involving altered IFN-α/β response gene transcription but not IFN-α/β pathway signaling. These results provide insight into the functioning of the IFN-α/β response in vivo and suggest that temperature elevation may represent an immune-enhancing therapeutic modality for a wide variety of IFN-α/β-sensitive infections and pathologies.

ABSTRACT Zika virus (ZIKV) is implicated in fetal stillbirth, microcephaly, intracranial calcifications, and ocular anomalies following vertical transmission from infected mothers. In adults, infection may trigger autoimmune inflammatory polyneuropathy. Transmission most commonly follows the bite of infected Aedes mosquitoes but may also occur through sexual intercourse or receipt of blood products. Definitive diagnosis through detection of viral RNA is possible in serum or plasma within 10 days of disease onset, in whole blood within 3 weeks of onset, and in semen for up to 3 months. Serological diagnosis is nonetheless critical because few patients have access to molecular diagnostics during the acute phase of infection and infection may be associated with only mild or inapparent disease that does not prompt molecular testing. Serological diagnosis is confounded by cross-reactivity of immune sera with other flaviviruses endemic in the areas where ZIKV has recently emerged. Accordingly, we built a high-density microarray comprising nonredundant 12-mer peptides that tile, with one-residue overlap, the proteomes of Zika, dengue, yellow fever, West Nile, Ilheus, Oropouche, and chikungunya viruses. Serological analysis enabled discovery of a ZIKV NS2B 20-residue peptide that had high sensitivity (96.0%) and specificity (95.9%) versus natural infection with or vaccination against dengue, chikungunya, yellow fever, West Nile, tick-borne encephalitis, or Japanese encephalitis virus in a microarray assay and an enzyme-linked immunosorbent assay (ELISA) of early-convalescent-phase sera (2 to 3 weeks after onset of symptomatic infection). IMPORTANCE The emergence of Zika virus (ZIKV) as a teratogen is a profound challenge to global public health. Molecular diagnosis of infection is straightforward during the 3-week period when patients are viremic. However, serological diagnosis thereafter of historical exposure has been confounded by cross-reactivity. Using high-density peptide arrays that tile the proteomes of a selection of flaviviruses to identify a ZIKV-specific peptide, we established two assays that enable sensitive and specific diagnosis of exposure to ZIKV. These assays may be useful in guiding clinical management of mothers at risk for potential exposure to ZIKV and enable insights into the epidemiology of ZIKV infections.

Koutango virus (KOUV), a close relative of West Nile virus, is highly neuroinvasive in animal models and has been associated with human disease. The complete genome of the KOUV prototype strain DakAnD5443 is reported here and may facilitate development of infectious clones for further characterization of this novel flavivirus., Competing Interests: The authors declare no conflict of interest.

ABSTRACT The small interfering RNA (siRNA) pathway is a major antiviral response in mosquitoes; however, another RNA interference pathway, the PIWI-interacting RNA (piRNA) pathway, has been suggested to be antiviral in mosquitoes. Piwi4 has been reported to be a key mediator of this response in mosquitoes, but it is not involved in the production of virus-specific piRNAs. Here, we show that Piwi4 associates with members of the antiviral exogenous siRNA pathway (Ago2 and Dcr2), as well as with proteins of the piRNA pathway (Ago3, Piwi5, and Piwi6) in an Aedes aegypti-derived cell line, Aag2. Analysis of small RNAs captured by Piwi4 revealed that it is predominantly associated with virus-specific siRNAs in Semliki Forest virus-infected cells and, to a lesser extent, with viral piRNAs. By using a Dcr2 knockout cell line, we showed directly that Ago2 lost its antiviral activity, as it was no longer bound to siRNAs, but Piwi4 retained its antiviral activity in the absence of the siRNA pathway. These results demonstrate a complex interaction between the siRNA and piRNA pathways in A. aegypti and identify Piwi4 as a noncanonical PIWI protein that interacts with members of the siRNA and piRNA pathways, and its antiviral activities may be independent of either pathway. IMPORTANCE Mosquitoes transmit several pathogenic viruses, for example, the chikungunya and Zika viruses. In mosquito cells, virus replication intermediates in the form of double-stranded RNA are cleaved by Dcr2 into 21-nucleotide-long siRNAs, which in turn are used by Ago2 to target the virus genome. A different class of virus-derived small RNAs, PIWI-interacting RNAs (piRNAs), have also been found in infected insect cells. These piRNAs are longer and are produced in a Dcr2-independent manner. The only known antiviral protein in the PIWI family is Piwi4, which is not involved in piRNA production. It is associated with key proteins of the siRNA and piRNA pathways, although its antiviral function is independent of their actions.

Hosts and viruses are locked in an evolutionary arms race. Hosts are constantly evolving to suppress virulence and replication, while viruses, which are reliant on host machinery for survival and reproduction, develop counterstrategies to escape this immune defense. Viruses must also adapt to novel conditions while establishing themselves in a host species. Both processes provide strong selection for viral adaptation. Understanding adaptive evolution in insect viruses can help us to better understand adaptive evolution in general and is important due to the use of these viruses as biocontrol agents and for protecting ecologically or economically important species from outbreaks. Here we examine the molecular evolution of baculoviruses and nudiviruses, a group of insect-infecting viruses with key roles in biocontrol. We looked for signatures of selection between genomes of baculoviruses infecting a range of species and within a population of baculoviruses. Both analyses found only a few strong signatures of positive selection, primarily in replication- and transcription-associated genes and several structural protein genes. In both analyses, we detected a conserved complex of genes, including the helicase gene, showing consistently high levels of adaptive evolution, suggesting that they may be key in antagonistic coevolution to escape host suppression. These genes are integral to the baculovirus life cycle and may be good focal genes for developing baculoviruses as effective biocontrol agents or for targeting baculoviruses infecting ecologically relevant species. Recombination and complex genomes make evolution in these double-stranded DNA viruses more efficient than that in smaller RNA viruses with error-prone replication, as seen via signatures of selection in specific genes within a population of baculoviruses. IMPORTANCE Most viral evolutionary studies focus on RNA viruses. While these viruses cause many human and animal diseases, such studies leave us with a lesser understanding of how DNA viruses adapt to hosts and how the host responds to these pathogens. In this paper, we focus on the evolution of baculoviruses, a group of insect-infecting DNA viruses, many of which have been used in biocontrol. We find that most of the genome is under purifying selection, with only a few key genes evolving adaptively. Our results provide a glimpse into how DNA viruses differ from RNA viruses in their evolutionary dynamics and identify genes that are key to DNA virus adaptation, improving our understanding of how this group of pathogens evolves. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) is a mosquito-transmitted flavivirus that can cause debilitating encephalitis. To delineate the mechanisms behind this pathology, we studied Ccr7-deficient mice, which afforded us the capacity to study infection in mice with disrupted peripheral cellular trafficking events. The loss of Ccr7 resulted in an immediate pan-leukocytosis that remained elevated throughout the infection. This leukocytosis resulted in a significant enhancement of leukocyte accumulation within the central nervous system (CNS). Despite an excess of virus-specific T cells in the CNS, Ccr7-deficient mice had significantly higher CNS viral loads and mortality rates than wild-type animals. Mechanistically, the elevated trafficking of infected myeloid cells into the brain in Ccr7-deficient mice resulted in increased levels of WNV in the CNS, thereby effectively contributing to neuroinflammation and lowering viral clearance. Combined, our experiments suggest that during WNV infection, Ccr7 is a gatekeeper for nonspecific viral transference to the brain. [ABSTRACT FROM AUTHOR]

Infected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: resistance, used to abrogate virus replication, and disease tolerance, used to avoid tissue damage without controlling viral burden. The principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of bluetongue disease of ruminants, as a model system to investigate the mechanisms of virus-host interactions correlating with disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Using primary cells from multiple donors, we show that BTV consistently reaches higher titers in ovine cells than cells from cattle. The variable replication kinetics of BTV in sheep and cow cells were mostly abolished by abrogating the cell type I interferon (IFN) response. We identified restriction factors blocking BTV replication, but both the sheep and cow orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells than cow cells, which results in an earlier downregulation of antiviral proteins. Moreover, by using RNA sequencing (RNA-seq), we also show a more pronounced expression of interferon-stimulated genes (ISGs) in BTV-infected cow cells than sheep cells. Our data provide a new perspective on how the type I IFN response in reservoir species can have overall positive effects on both virus and host evolution. IMPORTANCE The host immune response usually aims to inhibit virus replication in order to avoid cell damage and disease. In some cases, however, the infected host avoids the deleterious effects of infection despite high levels of viral replication. This strategy is known as disease tolerance, and it is used by animal reservoirs of some zoonotic viruses. Here, using a virus of ruminants (bluetongue virus [BTV]) as an experimental system, we dissected virus-host interactions in cells collected from species that are susceptible (sheep) or tolerant (cow) to disease. We show that (i) virus modulation of the host antiviral type I interferon (IFN) responses, (ii) viral replication kinetics, and (iii) virus-induced cell damage differ in tolerant and susceptible BTV-infected cells. Understanding the complex virus-host interactions in disease tolerance can allow us to disentangle the critical balance between protective and damaging host immune responses., Competing Interests: The authors declare no conflict of interest.