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MicroRNAs (miRNAs) are a group of small noncoding RNAs that regulate gene expression during important biological processes including development and pathogen defense in most living organisms. Presently, no miRNAs have been identified in the mosquito Culex tarsalis (Diptera: Culicidae), one of the most important vectors of West Nile virus (WNV) in North America. We used small RNA sequencing data and in vitro and in vivo experiments to identify and validate a repertoire of miRNAs in Cx. tarsalis mosquitoes. Using bioinformatic approaches we analyzed small RNA sequences from the Cx. tarsalis CT embryonic cell line to discover orthologs for 86 miRNAs. Consistent with other mosquitoes such as Aedes albopictus and Culex quinquefasciatus, miR-184 was found to be the most abundant miRNA in Cx. tarsalis. We also identified 20 novel miRNAs from the recently sequenced Cx. tarsalis genome, for a total of 106 miRNAs identified in this study. The presence of selected miRNAs was biologically validated in both the CT cell line and in adult Cx. tarsalis mosquitoes using RT–qPCR and sequencing. These results will open new avenues of research into the role of miRNAs in Cx. tarsalis biology, including development, metabolism, immunity, and pathogen infection.

Error-prone replication of RNA viruses generates the genetic diversity required for adaptation within rapidly changing environments. Thus, arthropod-borne virus (arbovirus) populations exist in nature as mutant swarms that are maintained between arthropods and vertebrates. Previous studies have demonstrated that West Nile virus (WNV) population dynamics are host dependent: In American crows, which experience extremely high viremia, purifying selection is weak and population diversity is high compared to American robins, which have 100 to 1000-fold lower viremia. WNV passed in robins experiences fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows higher genetic diversity within individual avian peripheral-blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a novel, barcoded version of WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes that each contained. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation. These studies further document the role of particular, ecologically relevant hosts in shaping virus population structure.Author SummaryWNV mutational diversity in vertebrates is species-dependent. In crows, low frequency variants are common, and viral populations are more diverse. In robins, fewer mutations become permanent fixtures of the overall viral population. We infected crows, robins and a chicken cell line with a genetically marked (barcoded) WNV. Higher levels of virus led to multiple unique WNV genomes infecting individual cells, even when a genotype was present at low levels in the input viral stock. Our findings suggest that higher levels of circulating virus in natural hosts allow less fit viruses to survive in RNA virus populations through complementation by more fit viruses. This is significant as it allows less represented and less fit viruses to be maintained at low levels until they potentially emerge when virus environments change. Overall our data reveal new insights on the relationships between host susceptibility to high viremia and virus evolution.

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are arthropod-borne viruses transmitted mainly by Aedes aegypti mosquitoes. These viruses have become endemic in large parts of North, Central, and South America. Arboviruses persistently infect mosquitoes throughout their life span and become infectious (i.e., expectorate infectious virus in saliva) after a period of time called the extrinsic incubation period (EIP). The duration of this infectiousness, however, is not well characterized. This is an important shortcoming because many epidemiological models assume that mosquitoes continue to be infectious for the duration of their life span. To define the duration of infectiousness for CHIKV and ZIKV, mosquitoes were infected orally with these viruses. Every 2 days, legs/wings, midguts, salivary glands, and saliva were collected from 30 to 60 mosquitoes and viral load measured. In CHIKV-infected mosquitoes, infectious virus in saliva peaked early (2-4 dpi), and then decreased rapidly and was rarely observed after 10 dpi. Viral RNA in infected tissues also decreased after the initial peak (4-8 dpi) but did so much less drastically. In ZIKV-infected mosquitoes, the infectious virus in saliva peaked at 12-14 dpi and dropped off only slightly after 14 dpi. In infected tissues, viral RNA increased early during infection, and then plateaued after 6-10 days. Our findings suggest that significant variation exists in the duration of the infectious period for arboviruses that is in part influenced by virus clearance from expectorated saliva.

Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C—40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of > 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimal conditions occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined fluctuating temperatures which better mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. Conversely, fluctuating temperature decreased vector competence. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas fluctuating temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology, including major effects on the selective environment within mosquitoes., Author summary Arthropod-borne viruses (arboviruses) have emerged in recent decades due to complex factors that include increases in international travel and trade, the breakdown of public health infrastructure, land use changes, and many others. Climate change also has the potential to shift the geographical ranges of arthropod vectors, consequently increasing the global risk of arbovirus infection. Changing temperatures may alter the virus-host interaction, ultimately resulting in the emergence of new viruses and virus genotypes in new areas. Therefore, we sought to characterize how temperature (both constant and fluctuating) alters the ability of Aedes aegypti and Aedes albopictus to transmit Zika virus, and how it influences virus populations within mosquitoes. We found that intermediate temperatures maximize virus transmission compared to more extreme and fluctuating temperatures. Constant temperatures increased positive selection on virus genomes, while fluctuating temperatures strengthened purifying selection. Our studies provide evidence that in addition to altering vector competence, temperature significantly influences natural selection within mosquitoes.

Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C - 40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of > 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimum occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics withinAedes aegyptiandAedes albopictusmosquitoes. We also examined diurnally fluctuating temperatures which more faithfully mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for bothAedesspecies. Transmission peaked at 10 days post-infection forAedes aegyptiand 14 days forAedes albopictus.The effect of diurnal temperature was distinct and could not have been predicted from constant temperature-derived data. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas diurnal temperatures led to strong purifying selection in bothAedesspecies. These findings demonstrate that temperature has multiple impacts on ZIKV biology within mosquitoes, including major effects on the selective environment within mosquitoes.Author SummaryArthropod-borne viruses (arboviruses) have emerged in recent decades due to complex factors that include increases in international travel and trade, the breakdown of public health infrastructure, land use changes, and many other factors. Climate change also has the potential to shift the geographical ranges of arthropod vectors, consequently increasing the global risk of arbovirus infection. Changing temperatures may also alter the virus-host interaction, ultimately resulting in the emergence of new viruses and virus genotypes in new areas. Therefore, we sought to characterize how temperature (both constant and fluctuating) alters the ability ofAedes aegyptiandAedes albopictusto transmit Zika virus, and how it influences virus populations within mosquitoes. We found that intermediate temperatures maximize virus transmission compared to more extreme and fluctuating temperatures. Constant temperatures increased positive selection on virus genomes, while fluctuating temperatures strengthened purifying selection. Our studies provide evidence that in addition to altering VC, temperature significantly influences the selective environment within mosquitoes.

The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses of humans. The interactions between DENVs and the human host that lead to asymptomatic, mild, or severe disease are poorly understood, in part, because laboratory models are poor surrogates for human DENV disease. Virologists are interested in how the properties of DENVs replicating in people compare with virions propagated on laboratory cell lines, which are widely used for research and vaccine development. Using clinical samples from a DENV type 1 epidemic in Sri Lanka and new ultrasensitive assays, we compared the properties of DENVs in human plasma and after one passage on laboratory cell lines. DENVs in plasma were 50- to 700-fold more infectious than cell culture-grown viruses. DENVs produced by laboratory cell lines were structurally immature and hypersensitive to neutralization by human antibodies compared with DENVs circulating in people. Human plasma and cell culture-derived virions had identical genome sequences, indicating that these phenotypic differences were due to the mature state of plasma virions. Several dengue vaccines are under development. Recent studies indicate that vaccine-induced antibodies that neutralized DENVs in cell culture assays were not sufficient for protecting people from DENV infections. Our results about structural differences between DENVs produced in humans versus cell lines may be key to understanding vaccine failure and developing better models for vaccine evaluation.

Arboviruses such as Zika virus (ZIKV, Flaviviridae; Flavivirus) replicate in both mammalian and insect hosts where they encounter a variety of distinct host defenses. To overcome these pressures, arboviruses exist as diverse populations of distinct genomes. However, transmission between hosts and replication within hosts can involve genetic bottlenecks, during which population size and viral diversity may be significantly reduced, potentially resulting in large fitness losses. Understanding the points at which bottlenecks exist during arbovirus transmission is critical to identifying targets for preventing transmission. To study these bottleneck effects, we constructed 4 “barcoded” ZIKV clones - 2 with an 8-base-pair degenerate insertion in the 3’ UTR and 2 with 8 or 9 degenerate synonymous changes in the coding sequence, theoretically containing thousands of variants each. We passaged these viruses 3 times each in 2 mammalian and 2 mosquito cell lines and characterized selection of the “barcode” populations using deep sequencing. Additionally, the viruses were used to feed three recently field-caught populations of Aedes aegypti mosquitoes to assess bottlenecks in a natural host. The barcoded viruses replicated well in multiple cell lines in vitro and in vivo in mosquitoes and could be characterized using next-generation sequencing. The stochastic nature of mosquito transmission was clearly shown by tracking individual barcodes in Ae. aegypti mosquitoes. Barcoded viruses provide an efficient method to examine bottlenecks during virus infection.AUTHOR SUMMARYIn general, mosquito-borne viruses like ZIKV must replicate in two very different host environments: an insect and a mammalian host. RNA viruses such as ZIKV must maintain genetic diversity in order to adapt to these changing conditions. During this transmission cycle, several barriers exist which can severely restrict viral genetic diversity, causing bottlenecks in the virus population. It is critical to understand these bottlenecks during virus transmission as this will provide important insights into the selective forces shaping arbovirus evolution within and between hots. Here, we employ a set of barcoded ZIKV constructs containing a degenerate stretch of nucleotides that can be tracked using next-generation sequencing. We found that the insertion site in the genome was an important determinant of the resulting diversity of the genetic barcode. We also found that bottlenecks varied between different mosquito populations and patterns of genetic diversity were distinct among individual mosquitoes within a single population, highlighting the randomness of virus dissemination in mosquitoes. Our study characterizes a new tool for tracking bottlenecks during virus transmission in vivo and highlights the importance of both viral and host factors on the maintenance of viral diversity.

Mosquito cell lines have been used extensively in research to isolate and propagate arthropod-borne viruses and understand virus-vector interactions. Despite their utility as an in vitro tool, these cell lines are poorly defined and may harbor insect-specific viruses. Accordingly, we screened four commonly-used mosquito cell lines, C6/36 and U4.4 cells from Aedes albopictus, Aag2 cells from Aedes aegypti, and Hsu cells from Culex quinquefasciatus, for the presence of adventitious (i.e. exogenous) viruses. All four cell lines stained positive for double-stranded RNA, indicative of RNA virus replication. We subsequently identified viruses infecting Aag2, U4.4 and Hsu cell lines using untargeted next-generation sequencing, but not C6/36 cells. PCR confirmation revealed that these sequences stem from active viral replication and/or integration into the cellular genome. Our results show that these commonly-used mosquito cell lines are persistently-infected with several viruses. This finding may be critical to interpreting data generated in these systems. Centers for Disease Control and Prevention [U50/CCU116806-01, U01/CK000509-01]; US Department of Agriculture Hatch Funds and Multistate Research Project [CONH00773, NE1443]; National Institute of Health, National Institute of Allergy and Infectious Diseases [AI067380, AI099042]; NIH/NCATS [UL1 TR001082] This work was supported in part by grants from the Centers for Disease Control and Prevention (U50/CCU116806-01 and U01/CK000509-01), the US Department of Agriculture Hatch Funds and Multistate Research Project (CONH00773 and NE1443), and the National Institute of Health, National Institute of Allergy and Infectious Diseases (AI067380, AI099042), and NIH/NCATS (UL1 TR001082).

Arthropod-borne viruses (arboviruses), such as Zika virus, chikungunya virus, and West Nile virus (WNV), pose continuous threats to emerge and cause large epidemics. Often, these events are associated with novel virus variants optimized for local transmission that first arise as minorities within a host. Thus, the conditions that regulate the frequency of intrahost variants are important determinants of emergence. Here, we describe the dynamics of WNV genetic diversity during its transmission cycle. By temporally sampling saliva from individual mosquitoes, we demonstrate that virus populations expectorated by mosquitoes are highly diverse and unique to each feeding episode. After transmission to birds, however, most genetic diversity is removed by strong purifying selection. Further, transmission of potentially mosquito-adaptive WNV variants is strongly influenced by genetic drift in mosquitoes. These results highlight the complex evolutionary forces a novel virus variant must overcome to alter infection phenotypes at the population level.

The magnitude and duration of vertebrate viremia is a critical determinant of arbovirus transmission, geographic spread, and disease severity. We find that multiple alphaviruses, including chikungunya (CHIKV), Ross River (RRV), and o’nyong ‘nyong (ONNV) viruses, are cleared from the circulation of mice by liver Kupffer cells, impeding viral dissemination. Clearance from the circulation was independent of natural antibodies or complement factor C3, and instead relied on scavenger receptor SR-A6 (MARCO). Remarkably, lysine to arginine substitutions at distinct residues within the E2 glycoproteins of CHIKV and ONNV (E2 K200R) as well as RRV (E2 K251R) allowed for escape from clearance and enhanced viremia and dissemination. Mutational analysis revealed that viral clearance from the circulation is strictly dependent on the presence of lysine at these positions. These findings reveal a previously unrecognized innate immune pathway that controls alphavirus viremia and dissemination in vertebrate hosts, ultimately influencing disease severity and likely transmission efficiency., eLife digest Viruses transmitted by blood-feeding insects, such as mosquitoes and ticks, cause serious human diseases. In recent years these viruses (also known as arboviruses) have re-emerged at an unprecedented scale, leading to global outbreaks of diseases such as Zika or chikungunya fever. The severity of these diseases and how easily they can be transmitted depends, in part, on the level of virus in the host’s bloodstream following infection. The more viral particles present in the blood, the easier it is for other insects that bite the host to become infected and help spread the disease. Yet, the mechanisms that hosts use to control the amount of virus present in the blood and how long it persists are poorly understood. To investigate this further, Carpentier et al. used a combination of molecular and genetic techniques to study how mice clear particles of arbovirus from their bloodstream. Surgically removing the spleen from infected mice revealed that this organ, which filters out unwanted or damaged materials from blood, is not required to clear some arbovirus particles. Carpentier et al. found that removing these arboviruses from the blood instead required Kupffer cells, a type of immune cell found in the liver. Genetically manipulating mice so they no longer produced a protein on the surface of Kupffer cells known as MARCO revealed that this receptor is needed to clear chikungunya viral particles. When MARCO was genetically deleted this led to an increase in the number of viral particles in the mice’s bloodstream, and allowed the virus to spread more rapidly throughout the bodies of the mice. Further experiments on three different types of arboviruses showed that in order to be cleared by MARCO, each of these viruses needed a lysine residue – one of the building blocks that makes up proteins – at defined positions within their protein sequence. These findings reveal a previously unknown mechanism for how hosts remove arbovirus particles from their bloodstream. Future studies could use this information to identify new ways to control the transmission and reduce the severity of these viral diseases.

Starting in 2013–2014, the Americas have experienced a massive outbreak of Zika virus (ZIKV) which has now reached at least 49 countries. Although most cases have occurred in South America and the Caribbean, imported and autochthonous cases have occurred in the United States. Aedes aegypti and Aedes albopictus mosquitoes are known vectors of ZIKV. Little is known about the potential for temperate Aedes mosquitoes to transmit ZIKV. Aedes vexans has a worldwide distribution, is highly abundant in particular localities, aggressively bites humans, and is a competent vector of several arboviruses. However, it is not clear whether Ae. vexans mosquitoes are competent to transmit ZIKV. To determine the vector competence of Ae. vexans for ZIKV, wild-caught mosquitoes were exposed to an infectious bloodmeal containing a ZIKV strain isolated during the current outbreak. Approximately 80% of 148 mosquitoes tested became infected by ZIKV, and approximately 5% transmitted infectious virus after 14 days of extrinsic incubation. These results establish that Ae. vexans are competent ZIKV vectors. Their relative importance as vectors (i.e., their vectorial capacity) depends on feeding behavior, longevity, and other factors that are likely to vary in ecologically distinct environments.

RNA interference is a crucial antiviral mechanism in arthropods, including in mosquito vectors of arthropod-borne viruses (arboviruses). Although the exogenous small interfering RNA (siRNA) pathway constitutes an efficient antiviral response in mosquitoes, virus-derived P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) have been implicated in the response to alpha-, bunya- and flaviviruses in Aedes spp. mosquitoes. Culex mosquitoes transmit several medically important viruses including West Nile virus (WNV), but are considerably less well studied than Aedes mosquitoes and little is known about antiviral RNA interference in Culex mosquitoes. Therefore, we sequenced small RNA (sRNA) libraries from different Culex cell lines and tissues infected with WNV. The clear majority of virus-derived sRNA reads were 21 nt siRNAs in all cell lines and tissues tested, with no evidence for a role of WNV-derived piRNAs. Additionally, we aligned sRNA reads from Culex quinquefasciatus Hsu cells to the insect-specific rhabdovirus, Merida virus, which persistently replicates in these cells. We found that a significant proportion of the sRNA response to Merida virus consisted of piRNAs. Since viral DNA forms have been implicated in siRNA and piRNA responses of Aedes spp. mosquitoes, we also tested for viral DNA forms in WNV infected Culex cells. We detected viral DNA in Culex tarsalis cells infected with WNV and, to a lesser amount, WNV and Merida virus-derived DNA in Culex quinquefasciatus Hsu cells. In conclusion, Hsu cells generated Merida virus-derived piRNAs, but our data suggests that the major sRNA response of Culex cells and mosquitoes to WNV infection is the exogenous siRNA response. It is also evident that sRNA responses differ significantly between specific virus-mosquito combinations. Future work using additional Culex-borne viruses may further elucidate how virus-derived piRNAs are generated in Culex cells and what role they may play in controlling replication of different viruses.

In urban settings, chikungunya, Zika, and dengue viruses are transmitted by Aedes aegypti mosquitoes. Since these viruses co-circulate in several regions, coinfection in humans and vectors may occur, and human coinfections have been frequently reported. Yet, little is known about the molecular aspects of virus interactions within hosts and how they contribute to arbovirus transmission dynamics. We have previously shown that Aedes aegypti exposed to chikungunya and Zika viruses in the same blood meal can become coinfected and transmit both viruses simultaneously. However, mosquitoes may also become coinfected by multiple, sequential feeds on single infected hosts. Therefore, we tested whether sequential infection with chikungunya and Zika viruses impacts mosquito vector competence. We exposed Ae. aegypti mosquitoes first to one virus and 7 days later to the other virus and compared infection, dissemination, and transmission rates between sequentially and single infected groups. We found that coinfection rates were high after sequential exposure and that mosquitoes were able to co-transmit both viruses. Surprisingly, chikungunya virus coinfection enhanced Zika virus transmission 7 days after the second blood meal. Our data demonstrate heterologous arbovirus synergism within mosquitoes, by unknown mechanisms, leading to enhancement of transmission under certain conditions.

Arboviruses such as West Nile, Zika, chikungunya, dengue, and yellow fever viruses have become highly significant global pathogens through unexpected, explosive outbreaks. While the rapid progression and frequency of recent arbovirus outbreaks is associated with long-term changes in human behavior (globalization, urbanization, climate change), there are direct mosquito–virus interactions which drive shifts in host range and alter virus transmission. This review summarizes how virus–mosquito interactions are critical for these viruses to become global pathogens at molecular, physiological, evolutionary, and epidemiological scales. Integrated proactive approaches are required in order to effectively manage the emergence of mosquito-borne arboviruses, which appears likely to continue into the indefinite future.

Background ZIKV is a new addition to the arboviruses circulating in the New World, with more than 1 million cases since its introduction in 2015. A growing number of studies have reported vector competence (VC) of Aedes mosquitoes from several areas of the world for ZIKV transmission. Some studies have used New World mosquitoes from disparate regions and concluded that these have a variable but relatively low competence for the Asian lineage of ZIKV. Methodology/Principal findings Ten Aedes aegypti (L) and three Ae. albopictus (Skuse) collections made in 2016 from throughout Mexico were analyzed for ZIKV (PRVABC59—Asian lineage) VC. Mexican Ae. aegypti had high rates of midgut infection (MIR), dissemination (DIR) and salivary gland infection (SGIR) but low to moderate transmission rates (TR). It is unclear whether this low TR was due to heritable salivary gland escape barriers or to underestimating the amount of virus in saliva due to the loss of virus during filtering and random losses on surfaces when working with small volumes. VC varied among collections, geographic regions and whether the collection was made north or south of the Neovolcanic axis (NVA). The four rates were consistently lower in northeastern Mexico, highest in collections along the Pacific coast and intermediate in the Yucatan. All rates were lowest north of the NVA. It was difficult to assess VC in Ae. albopictus because rates varied depending upon the number of generations in the laboratory. Conclusions/Significance Mexican Ae. aegypti and Ae. albopictus are competent vectors of ZIKV. There is however large variance in vector competence among geographic sites and regions. At 14 days post infection, TR varied from 8–51% in Ae. aegypti and from 2–26% in Ae. albopictus., Author summary Aedes aegypti is an efficient vector for arboviruses which is partially determined by its vector competence (VC) status, a highly variable trait. Based on previous reports, the VC of New World mosquitoes is limited for ZIKV. However, a VC assessment from additional geographical sources was lacking. Therefore, we evaluated the VC for ZIKV using recently colonized mosquitoes from Mexico. Aedes aegypti and Ae. albopictus were highly susceptible to ZIKV infection but varied greatly in their transmission rates. As with previous studies of other flaviviruses; VC for ZIKV was highly variable in both Ae. aegypti and Ae. albopictus but was greatest in Ae. aegypti, supporting its role as the main ZIKV vector.

Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require precise diagnostic tools that are also appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that distinguishes Zika viruses of Asian and African lineages. The assay does not detect chikungunya virus or flaviviruses such as dengue, yellow fever, or West Nile viruses. The assay conditions allowed direct detection of Zika virus RNA in cultured infected cells; in mosquitoes; in virus-spiked samples of human blood, plasma, saliva, urine, and semen; and in infected patient serum, plasma, and semen samples without the need for RNA isolation or reverse transcription. The assay offers rapid, specific, sensitive, and inexpensive detection of the Asian-lineage Zika virus strain that is currently circulating in the Western hemisphere, and can also detect the African-lineage Zika virus strain using separate, specific primers.

The recent emergence of both chikungunya and Zika viruses in the Americas has significantly expanded their distribution and has thus increased the possibility that individuals may become infected by more than one Aedes aegypti-borne virus at a time. Recent clinical data support an increase in the frequency of coinfection in human patients, raising the likelihood that mosquitoes could be exposed to multiple arboviruses during one feeding episode. The impact of coinfection on the ability of relevant vector species to transmit any of these viruses (that is, their vector competence) has not been determined. Thus, we here expose Ae. aegypti mosquitoes to chikungunya, dengue-2 or Zika viruses, both individually and as double and triple infections. Our results show that these mosquitoes can be infected with and can transmit all combinations of these viruses simultaneously. Importantly, infection, dissemination and transmission rates in mosquitoes are only mildly affected by coinfection., Several mosquito-transmitted viruses cocirculate in the Americas, but the potential for co-transmission is unknown. Here, Rückert et al. show that Aedes aegypti mosquitos have the potential to co-transmit chikungunya, dengue and Zika viruses and that coinfection does not overall affect dissemination or transmission rates.

Zika virus (ZIKV; family Flaviviridae , genus Flavivirus ) is a rapidly expanding global pathogen that has been associated with severe clinical manifestations, including devastating neurological disease in infants. There are currently no molecular clones of a New World ZIKV available that lack significant attenuation, hindering progress toward understanding determinants of transmission and pathogenesis. Here we report the development and characterization of a novel ZIKV reverse genetics system based on a 2015 isolate from Puerto Rico (PRVABC59). We generated a two-plasmid infectious clone system from which infectious virus was rescued that replicates in human and mosquito cells with growth kinetics representative of wild-type ZIKV. Infectious clone-derived virus initiated infection and transmission rates in Aedes aegypti mosquitoes comparable to those of the primary isolate and displayed similar pathogenesis in AG129 mice. This infectious clone system provides a valuable resource to the research community to explore ZIKV molecular biology, vaccine development, antiviral development, diagnostics, vector competence, and disease pathogenesis. IMPORTANCE ZIKV is a rapidly spreading mosquito-borne pathogen that has been linked to Guillain-Barré syndrome in adults and congenital microcephaly in developing fetuses and infants. ZIKV can also be sexually transmitted. The viral molecular determinants of any of these phenotypes are not well understood. There is no reverse genetics system available for the current epidemic virus that will allow researchers to study ZIKV immunity, develop novel vaccines, or develop antiviral drugs. Here we provide a novel infectious clone system generated from a recent ZIKV isolated from a patient infected in Puerto Rico. This infectious clone produces virus with in vitro and in vivo characteristics similar to those of the primary isolate, providing a critical tool to study ZIKV infection and disease.

In 2015, Zika virus (ZIKV; Flaviviridae; Flavivirus) emerged in the Americas, causing millions of infections in dozens of countries. The rapid spread of the virus and the association with disease outcomes such as Guillain-Barré syndrome and microcephaly make understanding transmission dynamics essential. Currently, there are no reports of vector competence (VC) of American mosquitoes for ZIKV isolates from the Americas. Further, it is not clear whether ZIKV strains from other genetic lineages can be transmitted by American Aedes aegypti populations, and whether the scope of the current epidemic is in part facilitated by viral factors such as enhanced replicative fitness or increased vector competence. Therefore, we characterized replication of three ZIKV strains, one from each of the three phylogenetic clades in several cell lines and assessed their abilities to be transmitted by Ae. aegypti mosquitoes. Additionally, laboratory colonies of different Culex spp. were infected with an American outbreak strain of ZIKV to assess VC. Replication rates were variable and depended on virus strain, cell line and MOI. African strains used in this study outcompeted the American strain in vitro in both mammalian and mosquito cell culture. West and East African strains of ZIKV tested here were more efficiently transmitted by Ae. aegypti from Mexico than was the currently circulating American strain of the Asian lineage. Long-established laboratory colonies of Culex mosquitoes were not efficient ZIKV vectors. These data demonstrate the capacity for additional ZIKV strains to infect and replicate in American Aedes mosquitoes and suggest that neither enhanced virus replicative fitness nor virus adaptation to local vector mosquitoes seems likely to explain the extent and intensity of ZIKV transmission in the Americas., Author Summary The mechanisms contributing to the explosive nature of the current ZIKV outbreak in the Americas are poorly understood. Therefore, we characterized the replication of three strains, one from each phylogenetic clade of ZIKV and evaluated virus strain differences in transmission efficiency by American mosquitoes. Our results suggest that the strain currently circulating in the Americas does not have unusually high infectivity for American Ae. aegypti as compared to the African strains used in this study. Colonized Culex mosquitoes were inefficient vectors. In vitro data suggested slower replication and decreased fitness for the currently circulating American strain compared to African strains isolated decades ago. Therefore, viral adaptation to local mosquitoes does not appear, at present, to be driving the current ZIKV outbreak in the Americas.

Ticks are vectors and reservoirs of many arboviruses pathogenic for humans or domestic animals; in addition, during bloodfeeding they can acquire and harbour pathogenic arboviruses normally transmitted by other arthropods such as mosquitoes. Tick cell and organ cultures provide convenient tools for propagation and study of arboviruses, both tick-borne and insect-borne, enabling elucidation of virus-tick cell interaction and yielding insight into the mechanisms behind vector competence and reservoir potential for different arbovirus species. The mosquito-borne zoonotic alphavirus Semliki Forest virus (SFV), which replicates well in tick cells, has been isolated from Rhipicephalus, Hyalomma, and Amblyomma spp. ticks removed from mammalian hosts in East Africa; however nothing is known about any possible role of ticks in SFV epidemiology. Here we present a light and electron microscopic study of SFV infecting cell lines and organ cultures derived from African Rhipicephalus spp. ticks. As well as demonstrating the applicability of these culture systems for studying virus-vector interactions, we provide preliminary evidence to support the hypothesis that SFV is not normally transmitted by ticks because the virus does not infect midgut cells.

Arthropod-borne RNA viruses exist within hosts as heterogeneous populations of viral variants and, as a result, possess great genetic plasticity. Understanding the micro-evolutionary forces shaping these viruses can provide insights into how they emerge, adapt, and persist in new and changing ecological niches. While considerable attention has been directed toward studying the population dynamics of mosquito-borne viruses, little is known about tick-borne virus populations. Therefore, using a mouse and Ixodes scapularis tick transmission model, we examined Powassan virus (POWV; Flaviviridae, Flavivirus) populations in and between both the vertebrate host and arthropod vector. We found that genetic bottlenecks, RNAi-mediated diversification, and selective constraints collectively influence POWV evolution. Together, our data provide a mechanistic explanation for the slow, long-term evolutionary trends of POWV, and suggest that all arthropod-borne viruses encounter similar selective pressures at the molecular level (i.e. RNAi), yet evolve much differently due to their unique rates and modes of transmission.

Although the Woman with the Skull relief has long been an established part of the canon, art historians have hitherto neglected to consider the architectural changes that were made over the centuries to the south transept portal of the cathedral of Santiago de Compostela, also called the Puerta de las Platerias. Most studies have assumed that the current position of the relief is identical with its original setting. Analysis of the architectural context and figurative style of the Woman with the Skull carving, however, makes clear that this extraordinary image was not originally intended for the south transept portal but must have been part of the Romanesque north portal, which was demolished in the eighteenth century. Called the Woman Taken in Adultery in the Pilgrim's Guide and represented as a seductive enchantress with long, flowing locks, the figure is stylistically and formally related to the relief panels of King David, the Creation of Adam, and Christ Bestowing Blessings. This essay will argue tha...

Progress toward understanding Zika virus (ZIKV) pathogenesis is hindered by lack of immunocompetent small animal models, in part because ZIKV fails to effectively antagonize Stat2-dependent interferon (IFN) responses in mice. To address this limitation, we first passaged an African ZIKV strain (ZIKV-Dak-41525) through Rag1(−/−) mice to obtain a mouse-adapted virus (ZIKV-Dak-MA) that was more virulent than ZIKV-Dak-41525 in mice treated with an anti-Ifnar1 antibody. A G18R substitution in NS4B was the genetic basis for the increased replication, and resulted in decreased IFN-β production, diminished IFN-stimulated gene expression, and the greater brain infection observed with ZIKV-Dak-MA. To generate a fully immunocompetent mouse model of ZIKV infection, human STAT2 was introduced into the mouse Stat2 locus (hSTAT2 KI). Subcutaneous inoculation of pregnant hSTAT2 KI mice with ZIKV-Dak-MA resulted in spread to the placenta and fetal brain. An immunocompetent mouse model of ZIKV infection may prove valuable for evaluating countermeasures to limit disease.

Langat virus (LGTV), one of the members of the tick-borne encephalitis virus (TBEV) complex, was firstly isolated from Ixodes granulatus ticks in Malaysia. However, the prevalence of LGTV in ticks in the region remains unknown. Surveillance for LGTV is therefore important and thus a tool for specific detection of LGTV is needed. In the present study, we developed a real-time quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) for rapid detection of LGTV. Our findings showed that the developed qRT-PCR could detect LGTV at a titre as low as 0.1 FFU/ml. The detection limit of the qRT-PCR assay at 95% probability was 0.28 FFU/ml as determined by probit analysis (p ≤ 0.05). Besides, the designed primers and probe did not amplify ORF of the E genes for some closely related and more pathogenic viruses including TBEV, Louping ill virus, Omsk hemorrhagic fever virus (OHFV), Alkhurma virus (ALKV), Kyasanur Forest Disease virus (KFDV) and Powassan virus (POWV) which showed the acceptable specificity of the developed assay. The sensitivity of the developed method also has been confirmed by determining the LGTV in infected tick cell line as well as LGTV- spiked tick tissues.

This is an open access article distributed under the terms of the Creative Commons Attribution License.-- et al., Tudor staphylococcal nuclease (Tudor-SN) and Argonaute (Ago) are conserved components of the basic RNA interference (RNAi) machinery with a variety of functions including immune response and gene regulation. The RNAi machinery has been characterized in tick vectors of human and animal diseases but information is not available on the role of Tudor- SN in tick RNAi and other cellular processes. Our hypothesis is that tick Tudor-SN is part of the RNAi machinery and may be involved in innate immune response and other cellular processes. To address this hypothesis, Ixodes scapularis and I. ricinus ticks and/or cell lines were used to annotate and characterize the role of Tudor-SN in dsRNA-mediated RNAi, immune response to infection with the rickettsia Anaplasma phagocytophilum and the flaviviruses TBEV or LGTV and tick feeding. The results showed that Tudor-SN is conserved in ticks and involved in dsRNA-mediated RNAi and tick feeding but not in defense against infection with the examined viral and rickettsial pathogens. The effect of Tudor-SN gene knockdown on tick feeding could be due to down-regulation of genes that are required for protein processing and blood digestion through a mechanism that may involve selective degradation of dsRNAs enriched in G:U pairs that form as a result of adenosine-to-inosine RNA editing. These results demonstrated that Tudor-SN plays a role in tick RNAi pathway and feeding but no strong evidence for a role in innate immune responses to pathogen infection was found., This research was supported by grants BFU2011-23896 and the European Union FP7 ANTIGONE project number 278976 (JdlF). NA was funded by Ministerio de Educacion y Ciencia, Spain. VN was funded by the European Social Fund and the Junta de Comunidades de Castilla-La Mancha (Program FSE 2007–2013), Spain. RS was supported by the project Postdok_BIOGLOBE (CZ.1.07/2.3.00/30.0032) and by the Grant 13-12816P (GA CR). OH was supported by the Grant Agency of the Czech Republic grant no. 13-27630P and European Union FP7 MODBIOLIN project number 316304. CR and LBS were supported by the United Kingdom Biotechnology and Biological Sciences Research Council's National Capability Grant to the Pirbright Institute.

Arthropod vectors, such as mosquitoes, ticks, biting midges and sand flies, transmit many viruses that can cause outbreaks of disease in humans and animals around the world. Arthropod vector species are invading new areas due to globalisation and environmental changes, and contact between exotic animal species, humans and arthropod vectors is increasing, bringing with it the regular emergence of new arboviruses. For future strategies to control arbovirus transmission, it is important to improve our understanding of virus-vector interactions. In the last decade knowledge of arthropod antiviral immunity has increased rapidly. RNAi has been proposed as the most important antiviral response in mosquitoes and it is likely to be the most important antiviral response in all arthropods. However, other newly-discovered antiviral strategies such as melanisation and the link between RNAi and the JAK/STAT pathway via the cytokine Vago have been characterised in the last few years. This review aims to summarise the most important and most recent advances made in arthropod antiviral immunity.

Zika virus (ZIKV) is an emerging flavivirus that causes congenital abnormalities and Guillain-Barré syndrome. ZIKV infection also results in severe eye disease characterized by optic neuritis, chorioretinal atrophy, and blindness in newborns and conjunctivitis and uveitis in adults. We evaluated ZIKV infection of the eye by using recently developed mouse models of pathogenesis. ZIKV-inoculated mice developed conjunctivitis, panuveitis, and infection of the cornea, iris, optic nerve, and ganglion and bipolar cells in the retina. This phenotype was independent of the entry receptors Axl or Mertk, given that Axl(-/-), Mertk(-/-), and Axl(-/-)Mertk(-/-) double knockout mice sustained levels of infection similar to those of control animals. We also detected abundant viral RNA in tears, suggesting that virus might be secreted from lacrimal glands or shed from the cornea. This model provides a foundation for studying ZIKV-induced ocular disease, defining mechanisms of viral persistence, and developing therapeutic approaches for viral infections of the eye.